Assay methods involving ouabain

ABSTRACT

Methods for diagnosing pre-hypertension, hypertension, congestive cardiomyopathy, renal failure, salt-sensitivity and adenomas and endocrine cell hyperplasias are disclosed. Also disclosed are methods for monitoring hypertension therapy, congestive cardiomyopathy therapy, renal failure therapy and adenoma and endocrine call hyperplasia therapy. These methods involve using an antibody having binding specificity to ouabain to immunologically measure the level of human ouabain in body fluid or tissue of a subject. Additionally, methods for treating a hypertensive subject by inducing passive or active immunity to human ouabain in the subject are disclosed, along with an antibody having binding specificity for ouabain.

The development of the present invention was supported by the UpjohnCompany and the University of Maryland at Baltimore.

FIELD OF THE INVENTION

The present invention relates to methods for diagnosingpre-hypertension, hypertension, congestive cardiomyopathy, renalfailure, salt-sensitivity and adenomas and endocrine cell hyperplasias.The present invention also relates to methods for monitoringhypertension therapy, congestive cardiomyopathy therapy, renal failuretherapy and adenoma and endocrine cell hyperplasia therapy. Thesemethods involve using an antibody having binding specificity to ouabainto immunologically measure the level of human ouabain in body fluid ortissue of a subject. Additionally, the present invention relates tomethods for treating a hypertensive subject by inducing passive oractive immunity to human ouabain in the subject, and to an antibodyhaving binding specificity for ouabain.

BACKGROUND OF THE INVENTION I. Endogenous Factors

Endogenous factors include a group of factors found in animal and humanbody fluids and tissues. These factors appear to be associated withnatriuresis, the excretion of sodium in the urine. Natriuresis arises asa result of inhibition of (Na⁺ +K⁺)ATPase (the biochemical equivalent ofthe sodium pump) by the endogenous factors. The sodium pump drives thetransepithelial resorption of sodium in animal and human kidneys (DeWardener, H.E. et al, Clin. Sci., 63:415-420 (1982)). However, (Na⁺+K⁺)ATPase is not limited to kidney tissue. That is, this enzyme can befound in virtually all animal and human cells.

There is extensive literature on the isolation and purification ofendogenous factors that inhibit (Na⁺ +K⁺)ATPase; the following is only apartial list: Gruber, K.A. et al, Nature, 287:743-745 (1980); Cloix,J.F. et al, Biochem. Biophys. Res. Commun., 131:1234-1240 (1985); Kelly,R.A. et al, J. Biol. Chem., 260:11396-11405 (1985); Fishman, M.C., Proc.Natl. Acad. Sci. USA, 76:4661-4663 (1979); Lichstein, D. et al, Biochem.Biophys. Res. Commun., 96:1518-1523 (1980); Akagawa, K., J. Neurochem.,42:775-780 (1984); Jandhyala, B.S. et al, Clin. Sci., 70:103-110 (1986);Carilli, C.T. et al, J. Biol. Chem., 260:1027-1031 (1985); Clarkson, E.et al, Kidney Internatl., 16:710-721 (1979); Kramer, H. et al, HormonalRegulation of Sodium Excretion, pp. 303-323 (1980); Cloix, J.F., FEBSLett., 176:223-228 (1984); de The, H. et al, J. Cardiovas. Pharmacol.,6:549-554 (1984); Devynck, M.A. et al, Clin. and Exper. Hyperten. -Theory and Pract., A6:441-453 (1984); and Tamura, M. et al, J.Hyperten., 5:219-255 (1981).

One such factor or family of factors, i.e., endogenous digitalis-likefactor or substance (hereinafter "EDLF" or "EDLS"), is believed to benatriuretic by virtue of its ability to inhibit (Na⁺ +K⁺)ATPase.

EDLF inhibits (Na⁺ +K⁺)ATPase in at least three different biochemicalassay systems (Hamlyn, J.M. et al, J. Biol. Chem., 264:7395-7404(1989)). However, EDLF does not cross-react with antibodies raisedagainst digoxin. In this regard, and in terms of chromatographic andkinetic properties, EDLF is different from some of the other previouslyisolated (Na⁺ +K⁺)ATPase inhibitors (Graves, S.W. Ann. Int. Med.,99:604-608 (1983); Gruber, K.A. et al, Nature, 287:743-745 (1980);Gruber, K.A. et al, Hyperten., 4:348-354 (1982); Vasdev, S. et al, Res.Commun. Chem. Pathol. Pharmacol., 49:387-399 (1985); Kramer, H.J. et al,Renal Physiol., 5:80-90 (1985); Kelly, R.A. et al, J. Biol. Chem.,260:11396-11405 (1985); and Crabos, M. et al, FEBS Lett., 176:223-228(1984)).

In at least two instances, digitalis-like factors with chromatographicproperties similar to EDLF have been described in bovine adrenal tissue(Tamura, M., Biochem., 27:4244-4253 (1988)); and in human urine (Goto,A. et al, Biochem. Biophys, Res. Commun., 152:322-327 (1988)). Theestimated molecular weights, as determined by fast atom bombardment massspectrometry, of these digitalis-like factors are 336 and 343 Daltons,respectively.

A digitalis-like factor has also been detected in human amniotic fluid(U.S. Pat. No. 4,780,314). The molecular weight of this factor isdescribed as being 150-250 Daltons. In addition, this factor is taughtto cross-react strongly with digoxin antibodies, is highly protein boundin plasma and is a substrate for catechol-o-methyl transferase (COMT).These properties distinguish this digitalis-like factor from the EDLFdescribed in the present invention, which is of higher molecular weight,does not cross-react with digoxin antibodies (Hamlyn, J.M. et al, J.Biol. Chem., 264:7395-7404 (1989)), is not highly protein bound inplasma and is not a substrate for COMT.

II. The Role of EDLF in Hypertension

It has been suggested that EDLF may play a pathogenetic role inhypertension through inhibition of (Na⁺ +K⁺)ATPase in vascular smoothmuscle (Blaustein, M.P., Am. J. Physiol., 323:C165-C173 (1977)).

In established hypertension, the plasma levels of EDLF appear to becorrelated with blood pressure (Hamlyn, J.M. et al, Nature, 300:650-562(1982); and Hamlyn, J.M. et al, J. Endocrinol., 122:409-420 (1989)).However, the levels of EDLF rise before the blood pressure does, as aresult of volume expansion (Hamlyn, J.M., J. Endocrinol., 122:409-420(1989)). The elevated levels of EDLF may therefore be used as apredictor of hypertension because EDLF increases vascular smooth musclecontractility, and thus apparently causes the elevation of the bloodpressure (Blaustein, M.P., Am. J. Physiol., 232:C165-C173 (1977); andBlaustein, M.P. et al, Japan. J. Hyperten., 11:107-117 (1989)).

Several types of hypertension, including essential hypertension,adrenocorticoid-induced hypertension (including primary aldosteronism)and pregnancy-induced hypertension, are the consequence of excessivesodium retention by the body. Initially, the salt (sodium and chloride)retention is manifested as an expansion of plasma volume (Hamlyn, J.M.et al, Am. J. Physiol., 251:F563-F575 (1986)). This expansion of plasmavolume, is chronic, is sufficient to cause the elevation of bloodpressure through an increase in vascular smooth muscle contractility andconsequent increase in peripheral vascular resistance (Guyton, A.C. etal, Am. J. Med., 52:584-594 (1972)). The volume expansion appears topromote the secretion of an EDLF that has been extracted from the plasmaof volume-expanded humans and purified by dialysis of human plasma,lyophilization of the dialysate, extraction of methanol-solublecomponents, and flash evaporation followed by preparative,semi-preparative and analytical scale reverse phase chromatography. Theresulting purified EDLF differs from the plant steroid ouabain(molecular weight of 585 Daltons) in that the rate of loss of inhibitoryactivity over the course of time in an environment of 6.0 N HCl at 110°C. is significantly greater for the purified EDLF than for ouabain.Further, under type II-phosphorylating conditions, this EDLF interactswith (Na⁺ +K⁺)ATPase with an affinity between 12-25 fold higher thanthat of ouabain (Hamlyn, J.M. et al, J. Biol. Chem., 264, 7394-7404(1989); and Hamlyn, J.M. et al, Hyperten., 13:681-689 (1989)). Thus,EDLF was not believed to be the plant steroid ouabain.

Moreover, the above-described purification procedures were subsequentlyfound by Applicants to produce an insufficient quantity of EDLF (<1.0μg) to carry out structural analysis. Further, attempts to scale up theprocedure to produce a sufficient quantity of EDLF (at least 10 μg)failed because of the tendency of the preparative column to becomecompletely blocked by the sample, resulting in a low yield. In addition,as a result of subsequent work (see Example 1 below), Applicantsdiscovered that the EDLF purified by the above-described procedures wasnot homogeneous, as had been reported.

III. Identification of EDLF A. Misidentification of EDLF

Antibodies against the plant steroid ouabain conjugated to bovine serumalbumin (hereinafter "BSA") have been raised and shown to be able todetect elevated levels of an unknown factor with "ouabain-likeimmunoreactivity" in the plasma of subjects with primary aldosteronism(Masugi, F., Biochem. Biophys. Res. Commun., 135:41-45 (1986)) and inthe plasma of subjects with essential hypertension (Masugi, F., Clin.Exper. Hyperten. - Theory Pract., A9:1233-1242 (1987)). This"ouabain-like immunoreactivity" was partially purified, and was found tobe an "unstable lipid", possibly "an unstable peroxide . . . of lowmolecular weight" (Masugi, F., J. Hyperten., 6:S351-S353 (1988)). Thus,the substance partially purified is not ouabain because ouabain is asteroid, not a lipid. Moreover, the results indicate that theanti-ouabain antibody employed is not highly specific for ouabain, andcross-reacts with substances in plasma other than cardenolides, i.e.,lipids, that may inhibit (Na⁺ +K⁺)ATPase. Thus, this antibody and assaymethod is not sufficiently selective, and therefore is not suitable fordetection of the EDLF that appears to elevate blood pressure inhypertensive subjects.

None of the other factors isolated to date have been reported tocross-react with antibodies raised against ouabain. In fact, it has beendemonstrated that the previously isolated factors do not cross-reactwith antibodies having binding specificity for ouabain (Kelly, R.A., J.Biol. Chem., 260:11396-11405 (1985)).

B. Correct Identification of EDLF

Using a newly developed procedure (see Example 1 below), Applicantshave, for the first time in the present invention, been able to isolateand purify EDLF so as to identify such. This new procedure involves theuse of hydrophobic chromatography employing an amberlite XAD-2 resin soas to enrich for EDLF prior to preparative chromatography, and then theuse of affinity chromatography employing (Na⁺ +K⁺)ATPase as the ligand.This type of affinity chromatography could not have been used to isolateand purify the EDLF described by Hamlyn, J.M. et al, J. Biol. Chem.,264:7395-7404 (1989) and Hamlyn, J.M. et al, Hyperten., 13:681-689(1989) because of the presence of numerous impurities which interactnon-selectively with (Na⁺ +K⁺)ATPase. Because of this non-selectiveinteraction, (Na⁺ +K⁺)ATPase was unable to bind EDLF with high affinity.

The present invention is based upon the discovery by Applicants, in thepresent invention, that EDLF is unique in that it cross-reacts 100%(within experimental error) with antibodies having binding specificityfor ouabain. Applicants have also purified EDLF to homogeneity in thepresent invention and have discovered that EDLF is indistinguishablefrom plant ouabain in terms of fast atom bombardment mass spectroscopyand biological properties; EDLF is thus a human ouabain. Ouabainheretofore was only thought to exist in plants.

It is believed, in the present invention, that human ouabain, atelevated levels, inhibits (Na⁺ +K⁺)ATPase in a variety of cells,including vascular smooth muscle cells and neurons, includingsympathetic neurons, that activate vascular smooth muscle. Inhibition of(Na⁺ +K⁺)ATPase is believed to raise intracellular sodium in variouscell types and lead to a secondary rise in intracellular calcium viasodium/calcium exchange (Bova, S. et al, Am, J. Physiol., 259:H409-H423(1990)). The increased availability of intracellular calcium in thearterial smooth muscle cells is believed to enhance contractility(Blaustein, M.P. et al, Ann, N.Y. Acad. Sci., 488:199-216 (1986);Woolfson, R. et al, Hyperten., 15:583-590 (1990); and Bova, S. et al,Am. J. Physiol., 259:H409-H423 (1990)), and eventually lead to a rise inblood pressure, when the ability of the cardiovascular reflexes tocontrol blood pressure is exceeded (Blaustein, M.P., Am. J. Physiol.,232:C165-173 (1977); and Blaustein, M.P. et al, Japan. J. Hyperten.,11:107-117 (1989)).

IV. Pre-Hypertension

Studies in pigs have suggested that EDLF may be increased immediatelyprior to the development of hypertension, i.e., at the pre-hypertensivephase (Hamlyn, J.M., J. Endocrin., 122:409-420 (1989)). Thus, thepresence of elevated levels of human ouabain in normotensive subjects isbelieved, in the present invention, to be indicative of thepre-hypertensive state and to represent a valuable indicator of futurerisk for development of hypertension.

V. White-Coat Hypertension

Some individuals have elevated levels of blood pressure, e.g., diastolicblood pressure about 90 to 104 mm Hg, on repeated visits to thephysician's office, but normal blood pressure during ambulatorymonitoring (Pickering, T.G. et al, J. Am. Med. Assn., 259:225-228(1988); and Rucker, L. et al, South. Med. J., 83:610-612 (1990)). Thepresence of normal levels of human ouabain in these subjects with highblood pressure is believed, in the present invention, to be indicativeof "white-coat hypertension" or a pseudo-hypertension state, and thus avaluable indication of subjects who do not need therapy forhypertension.

VI. Congestive Cardiomyopathy

Various types of cardiac dysfunction (cardiomyopathies) are associatedwith disturbances in the relationship between blood volume and arterialblood pressure. Examples of such cardiomyopathies include ischemic heartdisease, familial cardiomyopathy, alcoholic cardiomyopathy, peripartumcardiomyopathy, endocardial fibroelastosis, postcarditic cardiomyopathy,hypertensive cardiomyopathy, idiopathic cardiomyopathy, and varioussecondary forms of myocardial involvement, e.g., in connective tissuediseases and neuromuscular diseases, such as muscular dystrophy.

In instances where the cardiomyopathy involves hypocontractility of theleft or right heart or both, more blood returns to the heart via thevenous system than can be pumped away. The accumulation of fluid (saltand water) leads to congestion of the lungs and/or venous system. Theusual therapeutic regimen for cardiomyopathies consists of diuretics,sodium restriction and digitalis (digoxin) (Glick , G. et al, In:Harrision's Principles of Internal Medicine, 9th Ed., eds. Isselbacher,K. et al, McGraw Hill, New York, pp. 1141-1146 (1980)). It has beensuggested that part of the rationale for the effectiveness of digitalisin cardiomyopathy arises because this therapeutic agent compensates forthe absence or reduced levels (below normal) of an endogenous digitalis(Szent-Gyorgyi, A., In: Chemical Physiology of Contraction in Body andHeart Muscle, Academic Press, New York, pp. 86-91 (1953)). Thus, absenceor reduced levels of human ouabain is believed, in the presentinvention, to be indicative of future risk for developing congestivecardiomyopathy in otherwise normal subjects. In addition, reduced levelsof human ouabain in those subjects with congestive failure is believed,in the present invention, to identify those subjects within this groupwho are most likely to benefit from instigation of therapy using agents,such as digoxin and perhaps even human ouabain.

VII. Renal Failure

The kidney is responsible for the daily metabolism and "clearance" of alarge variety of compounds from the body. It is known that when ouabainis administered to a subject, the excretory function of the kidneyaccounts for removal of 50-80% of the amount of administered ouabain(Selden, R. et al, J. Pharmacol. Exp. Ther., 188:615-623 (1974); Lahrty,H. et al, Pharmacol. Clinica, 1:114-118 (1969); and Strobach, H. et al,Naunyn-Schmeideberg's Arch. Pharmacol., 334:496-500 (1986)). Thus,alterations in renal excretory function for human ouabain are believed,in the present invention, to be detectable by changes in the levels ofhuman ouabain. For example, a decrease in renal clearance of humanouabain is believed, in the present invention, to elevate levels ofhuman ouabain. Moreover, the degree to which levels of human ouabain areelevated is believed, in the present invention, to be proportional tothe degree of renal impairment. Thus, detection of an elevated level ofhuman ouabain is believed, in the present invention, to be indicative ofthose subjects with renal disease (indicated by above-normal levels ofserum creatinine) who may already have low-grade renal disease or whomay be at risk for future development of renal failure.

Patients with chronic renal failure are dependent upon dialysis for theremoval of unwanted or excess levels of a variety of compounds in thecirculation. The efficacy of dialysis can be monitored by measuringserum creatinine and other indicators, such as blood urea nitrogen andplasma potassium. Measurement of human ouabain is believed, in thepresent invention, to also represent a useful indicator of the efficacyof dialysis in these patients. The level of human ouabain followingdialysis and the rate at which the level of human ouabain is elevatedbetween dialysis are believed, in the present invention, to beindicative of the severity of renal failure and the rate at whichdisease may progress to complete renal failure.

VIII. Salt-Sensitivity

Salt-sensitivity has been defined as a significant increase in bloodpressure when a subject has an increased dietary intake of sodium fromlow (about 40 to 60 meq/24 hr) to high (about 200 to 250 meq/24 hr). Incontrast, the blood pressure of salt-insensitive persons does not changesignificantly with similar manipulations of dietary sodium intake(Kawasaki, T. et al, Am. J. Med., 64:193-198 (1978)).

Elevated levels of EDLF have been found in animal models of hypertensionassociated with sodium and water retention (Hamlyn, J.M., J. Endocrin.,122:409-420 (1989); and see Example 4 below). Based upon theseobservations, it is believed, in the present invention, that levels ofhuman ouabain in normotensive individuals are different in thoseindividuals who are slat-sensitive. Salt-sensitive individuals arebelieved, in the present invention, to have elevated levels of humanouabain because of the tendency of blood volume to be greater in theseindividuals at all levels of sodium intake. Thus, measurement of a 24-hrurinary sodium level (as an indicator of daily dietary sodium ingestion)when coupled with measurements of human ouabain is believed, in thepresent invention, to be indicative of the presence of salt-sensitivity.A salt-sensitive subject is believed, in the present invention, to havea significantly greater increase in the level of human ouabain thansalt-insensitive subjects when the dietary sodium intake is increasedfrom a low-salt to a high-salt diet.

The ability to define salt-sensitive subjects in the normotensive(pre-hypertensive) population is useful in the recommendation of dietaryand other therapeutic interventions which may prevent the futuredevelopment of hypertension in these susceptible subjects. In addition,the ability to diagnose salt-sensitivity in hypertensive subjects shouldbe similarly useful as an indicator of the response to different classesof anti-hypertensive agents. For example, salt-sensitive hypertensivesubjects with elevated levels of human ouabain would be expected to showa better anti-hypertensive response to diuretic agents than other typesof anti-hypertensive agents which do not act directly on blood volume.

IX. Adenomas and Endocrine Cell Hyperplasias

Adenomas are endocrine cell tumors, and endocrine cell hyperplasiasrefer to non-tumorous proliferation of these cells.

The secretion of human ouabain is believed, in the present invention, tobe elevated when the endocrine cells involved in the biosynthesis ofhuman ouabain become tumorous (adenomas) and oversecrete human ouabain.Similarly, hyperplasia of the endocrine cells involved in thebiosynthesis of human ouabain will lead to increased production andsecretion of human ouabain.

In addition, the level of human ouabain is believed, in the presentinvention, to be elevated in disorders associated with overproduction ofa variety of steroids which share the same clearance mechanism as humanouabain in the kidney. These disorders include adenomas, such asovarian, adrenal, pituitary and testicular adenomas and endocrine cellhyperplasias. Thus, the detection of elevated levels of human ouabain isbelieved, in the present invention, to indicate the existence of one ormore of these disorders.

Furthermore, the levels of human ouabain are believed, in the presentinvention, to be a useful indicator of the response of adenomas andendocrine cell hyperplasias to therapeutic agents and/or surgicalintervention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for diagnosingpre-hypertension.

A further object of the present invention is to provide a method fordiagnosing hypertension.

An additional object of the present invention is to provide a method fordiagnosing white-coat hypertension.

Another object of the present invention is to provide a method fordetermining subjects at risk to develop congestive cardiomyopathy.

Still another object of the present invention if to provide a method fordetermining subjects at risk to develop renal failure.

Yet another object of the present invention is to provide a method fordetermining the salt-sensitivity of subjects at risk to develophypertension and hypertensive subjects.

A further object of the present invention is to provide a method fordiagnosing adenomas or endocrine cell hyperplasias.

An additional object of the present invention is to provide a method formonitoring hypertensive therapy.

Another object of the present invention is to provide a method formonitoring congestive cardiomyopathy therapy.

Still another object of the present invention is to provide a method formonitoring renal failure therapy.

Yet another object of the present invention is to provide a method formonitoring adenoma and endocrine cell hyperplasia therapy.

A further object of the present invention is to provide a method fortreating hypertensive subjects by inducing passive immunity to ouabain.

An additional object of the present invention is to provide a method fortreating hypertensive subjects by inducing active immunity to ouabain.

Another object of the present invention is to provide an antibody havingbinding specificity for ouabain.

In a first embodiment, the above-described objects of the presentinvention have been met by a method for diagnosing pre-hypertensioncomprising the steps of:

(1) obtaining a body fluid or tissue sample from a subject with normalblood pressure;

(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain;

(3) using an antibody binding specificity for ouabain, immunologicallymeasuring the level of human ouabain in the resulting enriched bodyfluid or tissue sample of step (2); and

(4) comparing the level of human ouabain obtained in step (3) with anouabain standard so as to detect a normal or elevated level of humanouabain in the subject and to diagnose the absence or presence ofpre-hypertension, respectively.

In a second embodiment, the above-described objects of the presentinvention have been met by a method for diagnosing hypertensioncomprising the steps of:

(1) obtaining a body fluid or tissue sample from a subject with highblood pressure;

(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain;

(3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and

(4) comparing the level of human ouabain obtained in step (3) with anouabain standard so as to detect a normal or elevated level of humanouabain in the subject and to diagnose the absence or presence ofhypertension, respectively.

In a third embodiment of the present invention, the above-describedobjects have been met by a method for diagnosing white-coat hypertensioncomprising the steps of:

(1) obtaining a body fluid or tissue sample from a subject with highblood pressure;

(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain;

(3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and

(4) comparing the level of human ouabain obtained in step (3) with anouabain standard so as to detect a normal or elevated level of humanouabain in the subject and to diagnose the presence or absence ofwhite-coat hypertension, respectively.

In a fourth embodiment of the present invention, the above-describedobjects have been met by a method for determining subjects at risk todevelop congestive cardiomyopathy comprising the steps of:

(1) obtaining a body fluid or tissue sample from a subject withcardiomyopathy;

(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain;

(3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and

(4) comparing the level of human ouabain measured in step (3) with anouabain standard so as to detect a normal or reduced level of humanouabain in the subject and to diagnose the absence or presence of therisk of developing congestive cardiomyopathy, respectively.

In a fifth embodiment of the present invention, the above-describedobjects have been met by a method for determining subjects at risk todevelop renal failure comprising the steps of:

(1) obtaining a body fluid or tissue sample from a subject with renaldisease;

(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain;

(3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and

(4) comparing the level measured in (3) with an ouabain standard so asto detect a normal or elevated level of human ouabain in the subject andto diagnose the absence or presence of the risk of developing renalfailure, respectively.

In a sixth embodiment of the present invention, the above-describedobjects have been met by a method for determining the salt-sensitivityof a subject at risk to develop hypertension or a hypertensive subjectcomprising:

(1) obtaining a body fluid or tissue sample from a subject at risk todevelop hypertension or a hypertensive subject, respectively;

(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain;

(3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2);

(4) comparing the level of human ouabain measured in step (3) with anouabain standard so as to detect a normal or elevated level of humanouabain in the subject; and

(5) repeating steps (1)-(4) at suitable time intervals after theinitiation of a low-salt or high-salt diet and measuring the level ofhuman ouabain at each time interval so as to determine thesalt-sensitivity of said subject, wherein when the level of humanouabain is elevated and the elevated level of human ouabain is due to achange from a low-salt to a high-salt diet, such is indicative ofsalt-sensitivity.

In a seventh embodiment of the present invention, the above-describedobjects have been met by a method for diagnosing adenomas or endocrinecells hyperplasias comprising the steps of:

(1) obtaining a body fluid or tissue sample from a subject;

(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain;

(3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2);

(4) comparing the level of human ouabain measured in step (3) with anouabain standard so as to detect a normal or elevated level of humanouabain in the subject; and

(5) repeating steps (1)-(4) at suitable time intervals after theinitiation of a low-salt or high-salt diet and measuring the level ofhuman ouabain at each time interval so as to determine the presence ofadenomas or endocrine cell hyperplasias in said subject, wherein whenthe level of human ouabain is elevated and the elevated level of humanouabain is not due to a change from a low-salt to a high-salt diet, suchis indicative of adenomas or hyperplasias.

In a eighth embodiment, the above-described objects of the presentinvention have been met by a method for monitoring hypertension therapycomprising the steps of:

(1) obtaining a body fluid or tissue sample from a subject diagnosed asafflicted with hypertension;

(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain;

(3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and

(4) repeating steps (1)-(3) at suitable time intervals after theinitiation of hypertension therapy and measuring the level of humanouabain at each time interval so as to monitor the effect of saidhypertension therapy, wherein a reduced level of human ouabain over thecourse of hypertension therapy is indicative of effective hypertensiontherapy.

In a ninth embodiment, the above-described objects of the presentinvention have been met by a method for monitoring congestivecardiomyopathy therapy comprising the steps of:

(1) obtaining a body fluid or tissue sample from a subject diagnosed asafflicted with congestive cardiomyopathy;

(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain;

(3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and

(4) repeating steps (1)-(3) at suitable time intervals after theinitiation of congestive cardiomyopathy therapy and measuring the levelof human ouabain at each time interval so as to monitor the effect ofsaid congestive cardiomyopathy therapy, wherein an increasing level ofhuman ouabain over the course of congestive cardiomyopathy therapy isindicative of effective congestive cardiomyopathy therapy.

In a tenth embodiment, the above-described objects of the presentinvention have been met by a method for monitoring renal failure therapycomprising the steps of:

(1) obtaining a body fluid or tissue sample from a subject diagnosed asafflicted with renal failure:

(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain;

(3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and

(4) repeating steps (1)-(3) at suitable time intervals after theinitiation of renal failure therapy and measuring the level of humanouabain at each time interval so as to monitor the effect of said renalfailure therapy, wherein a decreasing level of human ouabain over thecourse of renal failure therapy is indicative of effective renal failuretherapy.

In a eleventh embodiment, the above-described objects of the presentinvention have been met by a method for monitoring adenoma or endocrinecell hyperplasia therapy comprising the steps of:

(1) obtaining a body fluid or tissue sample from a subject diagnosed asafflicted with adenomas or endocrine cell hyperplasias;

(2) enriching the resulting body fluid or tissues sample obtained instep (1) for human ouabain;

(3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and

(4) repeating steps (1)-(3) at suitable time intervals after theinitiation of adenoma or endocrine cell hyperplasia therapy andmeasuring the level of human ouabain at each time interval so as tomonitor the effect of said adenoma or endocrine cell hyperplasiatherapy, wherein a decreasing level of human ouabain over the course ofadenoma or endocrine cell hyperplasia therapy is indicative of effectiveadenoma or endocrine cell hyperplasia therapy.

In a twelfth embodiment of the present invention, the above-describedobjects have been met by a method for treating hypertensive subjectscomprising administering to a hypertensive subject a therapeuticallyeffective amount of an antibody having binding specificity for ouabainso as to induce passive immunity to human ouabain in said subject.

In a thirteenth embodiment of the present invention, the above-describedobjects have been met by a method for treating hypertensive subjectscomprising administering to a hypertension subject a therapeuticallyeffective amount of conjugates of ouabain so as to induce activeimmunity to human ouabain in said subject.

In a fourteenth embodiment of the present invention, the above-describedobjects have been met by an antibody having binding specificity forouabain.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, "pre-hypertension" means the presence of an elevatedlevel of human ouabain, but a normal level of blood pressure.

Normal blood pressure varies depending upon the age, height, weight, sexand race of the subject. Other factors include risks such as smoking,heredity and alcohol. Generally, normal blood pressure is considered torange from about 105/60 to 140/90 mm Hg, and typically is about 120/80mm Hg. Thus, a high (borderline) normal blood pressure would be greaterthan about 130/85 to less than about 140/90 mm Hg. Further, a high(above normal) blood pressure would be greater than about 140/90 mm Hg.

As used herein, "hypertension" means the presence of an elevated levelof human ouabain and an elevated level of blood pressure, i.e., abovenormal blood pressure.

Examples of hypertension include essential hypertension,adrenocorticoid-induced hypertension, pregnancy-induced hypertension,renal hypertension and pheochromocytoma.

As used herein, "white-coat hypertension" means the presence of a normallevel of human ouabain and an elevated level of blood pressure, i.e.,above normal blood pressure.

As used herein, "at risk to develop congestive cardiomyopathy" means thepresence of a reduced level of human ouabain and cardiomyopathy.

As used herein, "cardiomyopathy" means diseases due to dysfunction ofthe myocardium or cardiac muscle.

Examples of cardiomyopathy include ischemic heart disease, familialcardiomyopathy, alcoholic cardiomyopathy, peripartum cardiomyopathy,endocardial fibroelastosis, postcarditic cardiomyopathy, hypertensivecardiomyopathy, idiopathic cardiomyopathy, and various secondary formsof myocardial involvement, e.g., in connective tissue diseases andneuromuscular diseases, such as muscular dystrophy.

As used herein, "at risk to develop renal failure" means the presence ofan elevated level of human ouabain and renal disease.

As used herein, "renal disease" means diseases associated withdestruction of renal mass.

Examples of renal disease include glomerulonephritis, diabeticnephropathy, tubulointerstital disease, polycystic renal disease andnephrosclerosis. Elevated levels of serum creatinine, and perhaps alsoelevated levels of blood urea nitrogen, are known to be early signs ofrenal disease (Coe, F.L., In: Harrision's Principles of InternalMedicine, 9th Ed., eds. Isselbacher, K. et al, McGraw Hill, New York,pp. 215-219 (1980)).

As used herein "salt-sensitivity" means an elevated level of humanouabain as a result of a change from a low-salt to a high-salt diet.

Examples of "subjects at risk to develop hypertension" include thosesubjects who have one or both parents that are hypertensive, overweightsubjects, and subjects with high normal blood pressure and subjects withrenal disease.

As used herein "hypertensive subject" means a subject diagnosed asafflicted with hypertension.

As used herein "adenomas or endocrine cell hyperplasias" means anelevated level of human ouabain which is not a result of a change from alow-salt to a high-salt diet.

As used herein, "adenomas" refer to endocrine cell tumors, and"endocrine cell hyperplasias" refer to non-tumorous proliferation ofthese cells.

The adenomas and endocrine cell hyperplasias include ovarian, adrenal,pituitary or testicular adenomas and endocrine cell hyperplasias, aswell as adenomas and hyperplasias of human ouabain secreting endocrinecells.

A "low-salt diet" means an intake of about 60 meq of sodium per day orless, generally about 40 to 60 meq of sodium per day or greater,generally about 200 to 250 meq of sodium per day.

Whether or not a "low-salt diet" or a "high-salt diet" is being effectedcan be determined by measuring the daily urinary sodium excretion fromsaid subject. This is because essentially all of the daily salt intakeis excreted as sodium in the urine over a period of 24 hr. The dailyurinary sodium excretion from said subject is determined by collecting a24-hr urine specimen, measuring the total volume of the urine specimen,measuring the sodium concentration in an aliquot of the urine specimenusing conventional flame photometry techniques, and multiplying theconcentration in the aliquot by the total volume of the 24-hr urinespecimen (MacGregor, G.A et al, Lancet, I:351-355 (1982)).

The level of human ouabain in a healthy subject, i.e., one not afflictedwith pre-hypertension or hypertension, salt-sensitivity, cardiomyopathy,renal failure, adenomas or endocrine cell hyperplasias, will varydepending upon the body fluid and tissue which is obtained in step (1),and the age, weight, sex and race of the subject. Generally, a healthysubject has about 100 to 600 pmoles of human ouabain, more particularly,about 40 to 95 pmol/liter of human ouabain in its plasma, about 95 to600 pmol/liter of human ouabain in its urine, and about 10,000 to 30,000pmol/kg of human ouabain in its adrenal tissue.

The level of human ouabain in the plasma of a pre-hypertensive subject,hypertensive subject, a subject afflicted with congestivecardiomyopathy, a subject afflicted with renal failure and a subjectafflicted with adenomas and endocrine cell hyperplasias will varydepending on the age, weight, sex, and race of the subject.

The level of human ouabain in the plasma of a pre-hypertensive subjectis generally about 100 to 4000 pmol/liter.

The level of human ouabain in the plasma of a hypertensive subject isgenerally about 250 to 2000 pmol/liter.

The level of human ouabain in the plasma of a subject at risk to developcongestive cardiomyopathy is generally about 10 to 35 pmol/liter.

The level of human ouabain in the plasma of a subject at risk to developrenal failure is generally about 100 to 4000 pmol/liter.

In subjects at risk to develop hypertension, salt-sensitive subjects mayhave about a 2-fold or greater increases of human ouabain, typically a 2to 7-fold increase of human ouabain, whereas salt-insensitive subjectsmay have less than a 2-fold increase in human ouabain, when the dietarysodium intake is increased from a low-salt to a high-salt diet.

Alternatively, particularly in hypertensive subjects, who have elevatedlevels of human ouabain even on a low-salt diet, salt-sensitive subjectsmay have more than a 150 pmol/liter rise in the plasma level of humanouabain, typically a 150 to 400 pmol/liter rise in the plasma level ofhuman ouabain (generally a rise to a final level of about 250 to 2000pmol/liter of plasma), whereas salt-insensitive subjects may have lessthan a 150 pmol/liter rise in the plasma level of human ouabain, whenthe dietary sodium intake is increased from a low-salt to a high-saltdiet.

The level of human ouabain in the plasma of a subject afflicted withadenomas or endocrine cell hyperplasias is generally about 100 to 4000pmol/liter.

The body fluid obtained in step (1) is not critical to the presentinvention. Examples of the body fluid include plasma, cerebrospinalfluid, saliva, semen, sweat, urine and amniotic fluid. Plasma is thepreferred body fluid to be tested in the present invention because it isthe most convenient.

The tissue sample obtained in step (1) is not critical to the presentinvention. Examples of the tissue samples include adrenal tissue, redblood cells, lymphocytes, and platelets. Red blood cells are thepreferred tissue sample to be tested in the present invention.

It is preferable to employ body fluid in step (1) because the biologicalsignificance of human ouabain most likely arises from its presence inplasma, serum, etc.

The enrichment techniques employed in step (2) is not critical to thepresent invention.

The body fluid can be enriched for human ouabain by a solid phase methodas described in Example 3 below. Other methods of enrichment involving,for example, ultrafiltration, affinity chromatography, columnchromatography, trichloracetic acid, perchloric acid, and ammoniumsulfate, may be used. The solid phase method described in Example 3below to enrich plasma is the preferred method. In this procedure, polarcompounds, such as human ouabain, which interact with the solid phasecolumn, are separated from the bulk protein and salts. These lattermaterials would otherwise interfere with the antibody reaction. When thebulk protein and salts have been washed through the column, humanouabain is preferentially eluted by washing with 25% (v/v) acetonitrile.The acetonitrile wash is dried under vacuum, and resuspended in assaybuffer comprising 50 mM sodium phosphate buffer (pH 7.4) containing0.08% (w/v) NaCl, 0.5% (v/v) Tween 20, 11.4 mg/l thimerosal and 10 mg/mlof BSA (hereinafter "assay buffer") for use with the antibody havingbinding specificity for ouabain. The use of 25% (v/v) acetonitrile forremoving human ouabain from the column does not cause the elution oflipids from the column. Serum lipids are known to cause false positivereactions with antibodies directed against cardiac glycosides (Kelly,R.A. et al, J. Biol. Chem., 250:11396-11405 (1985); and Masugi, F., J.Hyperten., 6:S351-S353 (1988)). The enrichment protocol, therefore,constitutes a critical advantage in the selective measurement of humanouabain using antibodies having binding specificity for ouabain anddistinguishes the present invention from previous measurements ofouabain-like immunoreactivity in plasma (Masugi, F., Biochem. Biophys.Res. Commun., 135:41-45 (1986); and Masugi, F., Clin. Exper. Hyperten. -Theory Pract., A9:1233-1242 (1987)).

The tissue samples can be enriched by homogenizing it in 10 parts ofmethanol and centrifuging so as to obtain a high speed supernatant. Thesupernatant is dried under vacuum and the residue thoroughlyreconstituted in 10 parts of water containing 0.1% (w/v) trifluoroaceticacid (hereinfter "TFA"). Insoluble matter is removed by centrifugationand the supernatant applied to, e.g., a C₁₈ disposable column, asdescribed for plasma in Example 3 below.

As used herein, "an antibody having binding specificity for ouabain"means an antibody which has:

(1) high affinity for ouabain, i.e., a dissociation constant on theorder of about 7.0 nM or less of ouabain, generally about 0.5 to 7.0 nMof ouabain, typically about 5.0 nM of ouabain, and

(2) low cross-reactivity for the well-known steroids present in humanplasma, i.e., on the order of about 1.0% or less cross-reactivity,typically about 0.02 to 0.001% cross-reactivity.

The antibody can be polyclonal or monoclonal in nature. Polyclonalantibodies can be reproducibly prepared as described in Example 2 below.

The antibody of the present invention differs from the known antibodieswhich have been raised against ouabain due to the nature of the ouabainconjugates used to prepare the antibodies. That is, high antibody titers(>10⁶) are obtained by employing sequential immunization and boosts withAntigens 1, 2 and 3 described in Example 2 below. These antigens wereused so as to direct the immunological response to ouabain and not tothe conjugate or the linker. This ensured the production of an antibodyhighly specific to ouabain. Previous attempts to raise ouabainantibodies have used only a single conjugate of ouabain and BSA forinitial immunization and subsequent boosters (Smith, T., J. Clin.Invest., 51:1583-1593 (1972); and Masugi, F., Biochem. Biophys. Res.Commun., 135:41-45 (1986)).

The level of human ouabain in the extracted body fluid or tissue sampleis immunologically measured using any conventional immunologicaltechnique, such as enzyme-linked immunosorbent assay (hereinafter"ELISA") or radioimmunoassay (hereinafter "RIA") (Chard, T., AnIntroduction to Radioimmunoassay and Related Techniques, Elsevier,Amsterdam (1987)). An ELISA is the preferred immunological techniquewhen rapid results with large numbers of samples may be desired,although an RIA may be preferred for high precision measurements.

It should be apparent to one skilled in the art that the assayconditions set forth in Example 3 are preferred to optimize the assayfor sensitivity and minimize the errors that may result from the assayof body fluids and tissue samples of slightly varying composition.However, it will also be recognized by one skilled in the art that, aslong as the unknown samples and ouabain standard are assayed undersubstantially the same conditions, the method of this invention can bepracticed in all of its aspects.

Ouabain used in the ouabain standard is commercially available from, forexamples, Sigma Chemical Company (St. Louis, Mo.), Calbiochem (La Jolla,Calif.), Aldrich (Milwaukee, Wis.).

Alternative, the ouabain used in the ouabain standard can be prepared asdescribed by Schwartze, E.W. et al, J. Pharmacol. Exp. Therap.,36:481-491 (1929).

When determining salt-sensitivity, diagnosing adenomas and endocrinecell hyperplasias or monitoring therapy, a suitable time interval formeasuring levels of human ouabain in body fluids or tissue samples maybe daily or weekly or any other suitable interval. A daily interval maybe preferable for determining salt-sensitivity or diagnosing adenomasand endocrine cell hyperplasias because changes in the level of humanouabain in the subject in response to changes in dietary salt intake arebelieved, in the present invention, to be very rapid. On the other hand,a weekly interval may be preferable for monitoring changes in the levelof human ouabain in the subject in response to pharmacologic therapybecause changes in the level of human ouabain in the subject in responseto pharmacologic therapy are believed, in the present invention, to berelatively slow.

As used herein, "hypertension therapy" includes the used of a low-saltdiet, a diuretic, and the use of other pharmacological andnon-pharmacological treatments, such as exercise, cessation of smoking,cessation of alcohol intake, and administration of an α-blocker, aβ-blocker or other sympatholytic agent, a converting enzyme inhibitor, acalcium channel blocker, or a combination thereof. These therapies arewell known in the art (Kaplan, N., Clin. Hyperten., 4th Ed., William &William, Baltimore, pp. 147-272 (1986)).

As used herein, "congestive cardiomyopathy therapy" includes the use ofa low-salt diet, and administration of a diuretic agent, a cardiotonicsteroid, such as digoxin, a vasodilator, a converting enzyme inhibitor,a phosphodiesterase inhibitor, or a combination thereof. These therapiesare well known in the art (Braunwald, E., In: Harrison's Principles ofInternal Medicine, 9th Ed., eds. Isselbacher, K.J. et al, McGraw-Hill,New York, pp. 1040-1044 (1980)).

As used herein, "renal failure therapy" includes the use of dietarypotassium restriction, dietary protein restriction and dialysis. Thesetherapies are well known in the art (Braunwald, E., In: Harrison'sPrinciples of Internal Medicine, 9th Ed., eds. Isselbacher, K.J. et al,McGraw-Hill, New York, pp. 1040-1044 (1980); and Brenner, B.M., In:Harrison's Principles of Internal Medicine, 9th Ed., eds. Isselbacher,K.J. et al, McGraw-Hill, New York, pp. 1305-1306 (1980)).

As used herein, "adenoma or endocrine cell hyperplasis therapy" includesthe use of spironolactone or surgery. These therapies are well known inthe art (Williams, G.H. et al, In: Harrison's Principles of InternalMedicine, 9th Ed., eds. Isselbacher, K.J. et al, McGraw-Hill, New York,pp. 1723-1726 (1980); and Kohler, P.O., In: Harrison's Principles ofInternal Medicine, 9th Ed., eds. Isselbacher, K.J. et al, McGraw-Hill,New York, pp. 1679-1680 (1980)).

In a method for treating hypertension of the present invention, theantibody having binding specificity for ouabain is administered to thesubject to as to bring about passive immunity. In this method, a subjectthat has an elevated level of ouabain is administered antibodies whichhave binding specificity for ouabain. These antibodies will bind to freehuman ouabain in the body fluid, and thus prevent inhibition of (Na⁺+K⁺)ATPase by human ouabain, resulting in a lowering of blood pressure.

The amount of antibody to administer will vary depending upon the age,weight and sex of the subject. Generally, the dosage to be administeredis in the range of about 0.5 to 20 μg of antibody per kg of body weight,preferably about 2.0 to 3.0 μg of antibody per kg of body weight.

The antibody will generally be administered intravenously as a slowinfusion, except in situations where blood pressure is elevated to alift-threatening level. Under such emergency conditions, rapidintravenous injections may be given.

The method of antibody production described herein can also be used toproduce active immunity in subjects with ouabain-dependent hypertension.Such subjects would be immunized using, e.g., Conjugates 1, 2 and 3described in Example 2 below.

More specifically, an emulsion containing about 1.0 to 30 mg of any oneof Conjugates 1, 2 or 3 in 0.5 ml saline and 0.5 ml of Freund's completeadjuvant would be injected subcutaneously into the upper arm. About 4 to6 weeks after the initial immunization, a boost consisting of about 1.0to 30 mg of any one of the remaining Conjugates in 0.5 ml saline and 0.5ml Freund's incomplete adjuvant would be injected subcutaneously in theupper arm. Then, an optional boost consisting of about 1.0 to 30 mg ofthe remaining Conjugate in 0.5 ml saline and 0.5 ml Freund's incompleteadjuvant would be injected subcutaneously in the upper arm about 4 to 6weeks later. Then, optionally, a final boost consisting of about 1.0 to30 mg of any one of Conjugates 1, 2 or 3 in 0.5 ml saline and 0.5 mlFreund's incomplete adjuvant would be injected subcutaneously in theupper arm about 4 to 6 weeks later.

The nature of the combined carrier-linking agent is unique to Conjugates1-3 so as to ensure that the antisera response will be directed againstouabain and not the carrier or linking agent. That is, the carrier andlinking agent vary among Conjugates 1-3. The use of a combination ofdifferent conjugates (containing different carriers and linking agents)is important for preparing antibodies having binding specificity forouabain. However, it is not critical to specifically use Conjugates 1-3.Other conjugates (containing other carriers and/or linking agents) canbe readily developed by one skilled in the art, and when employed incombination, used to prepare antibodies having binding specificity forouabain.

The carriers and linking agents employed are not critical to the presentinvention (Hurn, B.A.L. et al, Methods Enzymol., 70:104-142 (1980);Naegele, W. et al, In: Radioimmunoassay of Steroid Hormones, 2nd Ed.,ed. Gupta, D., Verlag Chemie, Weinheim (Fed. Rep. Germany), pp. 55-72(1980); and Chard, T., An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier, Amsterdam, pp. 93-97 (1987)). Examples of carrierswhich can be employed include BSA, ovalbumin, polylysine, thyroglobulin,keyhole limpet hemocyanin and equine serum albumin. Examples of linkingagents which can be employed include any reagent which provides a freeamine after linkage to the carrier such as any alkyl diamine, such ashexane diamine; any dihydrazide, such as succinyldihydrazide; or anyamino acid-N-carbopxyanhydride, such as alanine-N-carboxyanhydride.

In addition to their use in the assays described above, the antibodiesdescribed herein are useful either alone or when conjugated to a matrixfor extraction and purification of human ouabain from body fluids andtissues.

The following examples are provided for illustrative purposes only andare in no way intended to limit the scope of the present invention.

In the examples below, all solutions were made up in glass-distilledwater and the water referred to in the examples was glass-distilled.

EXAMPLE 1 Purification and Characterization of Human Ouabain I.Purification of Human Ouabain

Approximately 85 liters of human plasma were obtained from 30 donorsirrespective of age, sex and blood pressure and dialyzed at 23° C. for24 hr against 3 volumes of 10 mM ammonium acetate (pH 6.8) (Hamlyn, J.M.et al, J. Biol. Chem., 264:7395-7404 (1989)).

The resulting dialysate was split into 4 batches and applied to anAmberlite XAD-2 column (bed volume =3.5 liters) previously washed with10 liters of water. The adsorbed materials were eluted with 1 volume ofmethanol and dried under vacuum at 50° C. The solids were combined into2 batches, resuspended in water and fractionated by HPLC on apreparative-scale, C₁₈ reverse-phase column. The column was eluted witha 0-100% (v/v) acetonitrile gradient containing 1.0% (w/v) TFA over 180min (Hamlyn, J.M. et al, J. Biol. Chem., 264:7395-7404 (1989)).

Fractions from the column were assayed for inhibition ofouabain-sensitive ⁸⁶ Rb uptake by washed human red blood cells obtainedby venipuncture. This assay was used as an index of sodium transportactivity (Hamlyn, J.M. et al, J. Biol. Chem., 264:7395-7404 (1989)).

More specifically, freshly washed cells were incubated for 2 hr at 37°C. (16% final hematocrit) in a final volume of 150 μl of flux buffercomprising 150 mM NaCl, 2.0 mM BrCl containing tracer amounts of ⁸⁶ Rb(DuPont--New England Nuclesr), 1.0 mM MgCl₂, 1.0 mM Na₂ HPO₄, 2.0 mMCaCl₂, 5.0 mM glucose and 20 mM HEPES-Tris (pH 7.4) (hereinafter "fluxfubber"). Incubations were initiated by addition of column fractions tothe cells. Aliquots were removed at various times and mixed withice-cold flux buffer lacking ⁸⁶ Rb to quench the reactions. The quenchedreactions were applied to a silicone oil cushion (Hysol XF1792B, DexterCorp., Olean, N.Y.) in microcentrifuge tubes and centrifuged at 800×gfor 10 min at 4° C. The red cell pellet was cut from deep-frozen tubesand trapped ⁸⁶ Rb was determined by γ-counting. Flux data were expressedas nmol of Rb uptake/10⁸ cells. In each case, a value of 1.11×10¹³cells/liter was used to convert packed cell volumes to cell number forthe flux data calculations. Ouabain-insensitive ⁸⁶ Rb uptake wasestimated in parallel experiments by inclusion of 1.0 mM ouabain in theflux buffer. Ouabain-sensitive ⁸⁶ Rb uptake was taken as the totaluptake minus that obtained in the presence of 1.0 mM ouabain. Thefractions found to inhibit ouabain-sensitive ⁸⁶ Rb uptake were thenassayed for the ability to inhibit (Na⁺ +K⁺)ATPase.

More specifically, (Na⁺ +K⁺)ATPase was purified from the outer medullaof dog kidney using the angle rotor procedure (Jorgensen, P.L., Biochim.Biophys. Acta, 356:36-52 (1974)) and stored frozen at -60° C. at 1.0mg/ml in 25 mM imidazole-HCl (pH 7.5) and 1.0 mM EDTA, in smallaliquots. (Na⁺ +K⁺)ATPase activity was determined at 37° C. from ratemeasurements in 1.0 ml of coupled optical assay mixture comprising 20 mMKCl, 100 mM NaCl, 4.5 mM MgSO₄, 5.0 mM EGTA, 3.0 mM ATP-Na₂, 1.2 mMphosphoenol-pyruvate-tricyclohexylammonium, 0.3 mM NADH, 100 mM TES-Tris(ph 7.4), 5.0 units of lactate dehydrogenase, and 5.0 units of pyruvatekinase (Hamlyn, J.M. et al, Nature, 300:650-652 (1982)). (Na⁺ +K⁺)ATPaseactivity was 99% ouabain-inhibitable with activities of 10-18μmol/min/mg of membrane protein. Maximally effective concentrations ofdigitalis-like activity had no effect on the enzymatic components of thecoupled optical assay as determined by inspection of rates in responseto addition of 20-50 μM ADP. The maximal capacity of the coupled opticalassay was at least 500-2000 fold greater than the typical maximalhydrolytic rate of the (Na⁺ +K⁺)ATPase and was not rate limiting underany assay condition described herein.

The protein concentration was estimated by dye binding (Bradford, N.Mex., Anal. Biochem., 72:248-254 (1976)) using dried BSA as standard.

(Na⁺ +K⁺)ATPase inhibitory material eluting between 84 and 88 min(approximately 20% (v/v) acetonitrile) was combined to form a singlefraction and then subjected to affinity extraction with partiallypurified lamb kidney (Na⁺ +K⁺)ATPase (Lane, L.K. et al, Prep Biochem.,9:157-170 (1979)).

More specifically, the single fraction was incubated for 3 hr at 37° C.in Tris buffer comprising 200 mM Tris-HCl (pH 7.2), 5.0 mM MgCl₂ and 5.0mM NaH₂ PO₄ /Na₂ HPO₄ (hereinafter "Tris buffer") to which anapproximate 2-fold molar excess of (Na⁺ +K⁺)ATPase was added. Underthese conditions, a variety of cardenolides bind with high affinity to(Na⁺ +K⁺)ATPase. Following incubation, the reaction mixture wascentrifuged at 150,000 ×g for 2 hr at 4° C. to separate enzyme andenzyme-inhibitor complexes from soluble, unbound substances. The pelletwas washed twice with Tris buffer, and recentrifuged as described above.Then, the pellet was resuspended in buffer comprising 2.0 mM Tris-HCl(pH 7.2) and 5.0 mM EDTA-Tris, and incubated for 6 hr at 37° C. toinduce dissociation of enzyme-inhibitor complexes. Next, the enzyme waspelleted by centrifugation as described above and the supernatant waslyophilized. The lyophilized solids were taken up in water and, afterfiltration through a 5.0 μm filter (Acrodisc, Gelman Sciences Inc., AnnArbor, Mich.), subjected to 2 sequential HPLC steps: (1) a Waterssemi-preparative phenyl column (Milipore Corp., Milford, Mass.), elutedwith a 0 to 10% (v/v) isopropanol gradient over a period of 0 to 5 min,and then eluted with a 10 to 30% (v/v) isopropanol gradient over aperiod of 5 to 55 min; and (2) a Beckman semi-preparative C₁₈ column(Beckman, Palo Alto, Calif.), eluted with a 0 to 10% (v/v) acetonitrilegradient over a period of from 0 to 5 min, and then eluted with a 10 to30% (v/v) acetonitrile gradient over a period of 5 to 55 min.

A single peak of (Na⁺ +K⁺)ATPase inhibiting material, corresponding to12 μg of human ouabain, was associated with a narrow symmetrical UVabsorbing peak with no leading or trailing edges. 50 ml of plasmaequivalents from the UV peak at 32 min inhibited ouabain-sensitive ⁸⁶ Rbuptake by 80% with no effect on ouabain-insensitive uptake.

The overall purification was calculated to be >10¹⁰ -fold with respectto the starting plasma (dry weight). The minimum calculated plasmaconcentration of human ouabain in the donor plasma was 321 pM aftercorrection for losses in dialysis (25%). Chromtographic losses were notdetermined.

II. Structural Characterization of Human Ouabain

Purified human ouabain was analyzed by fast atom bombardment massspectrometry (FAB MS) using a VG 70 SE double focusing mass spectrometer(VG Instruments, Manchester, England).

More specifically, FAB spectra of the biologically active fraction andthe two adjacent fractions (dissolved in a glycerol/thioglycerol matrix)from the final purification step of Section I. above were acquired overa mass range of m/z 100 to m/z 2500 Daltons. A unique protonatedmolecular ion at m/z 585 was observed in the active fraction. Anaccurate mass of 585.295 Daltons for human ouabain was determined insubsequent FAB MS by peak matching the m/z 585 peak with glycerolcluster ions. Based upon this determination, an elemental composition ofC₂₉ H₄₅ O₁₂ was predicted for human ouabain. This composition matchesthat for ouabain (calculated accurate mass 585.291 for the protonatedspecies). Additional experiments, using linked scan MS/MS were used toanalyze the daughter ion fragment of the m/z 585 peak for human ouabainand ouabain. Both spectra were identical and showed a major ion at m/z439 corresponding to the aglycone of ouabain. Thus, the sugar moiety ofhuman ouabain, like that of ouabain, is a deoxyhexose.

Subsequently, actylated derivatives of human ouabain and ouabain wereexamined by FAB MS.

FAB spectra were acquired over the mass range of from m/z 100 to m/z1500 using a VG 70 SE mass spectrometer equipped with a Cs ion gun.Samples of ouabain and human ouabain were acetylated with 50 μl ofacetic anhydride/15 mg/ml of dimethyl aminophyridine (DMAP) in phyridine(1:1) for 2 hr at room temperature. After excess reagent was removedunder vacuum, the samples were dissolved in 0.5 μl ofglycerol/thioglycerol (1:1).

After acetylation, the protonated molecular ions at m/z 585 disappearedand several new ions at higher masses were present. The new peaks wereinterpreted as follows: m/z 958, [584]+6 acetyl group (Ac) + Na⁺ ; m/z901, [584]+7 Ac +Na⁺ ; m/z 959, [584] 6 Ac +DMAP +H⁺ ;m/z 1001, [584]+7Ac +DMAP +H⁺ ; m/z 1043, [584]+8 Ac+DMAP +H⁺.

The resultant spectra for human ouabain and ouabain are identical. Bothshow abundant ions corresponding to the addition of six acetyl groupsand less abundant ions for species with 7 and 8 acetyl groups. Theseresults are consistent with the structure of ouabain which has sixprimary and secondary OH groups and two hindered tertiary OH groups.

The structure of ouabain is shown below. ##STR1##

The accurate mass data, identical daughter ion and derivative spectrafollowing acetylation suggest that human ouabain and ouabain areisomeric if not identical compounds.

III. Biological Characterization of Human Ouabain

Dose-response curves for human ouabain were generated for (1) inhibitionof ouabain-sensitive uptake of ⁸⁶ Rb, (2) competition for ³ H-ouabainbinding, (3) inhibition of (Na⁺ +K⁺)ATPase activity, and (4) competitionfor binding to antibodies having binding specificity for ouabain(Hamlyn, J.M. et al, J. Biol. Chem., 264:7395-7404 (1989)).

A. Inhibition of Ouabain-sensitive Uptake of ⁸⁶ Rb

The inhibition of ouabain-sensitive uptake of ⁸⁶ Rb was carried out asdescribed above. The calculated apparent K_(d) value (and Hillcoefficient) for human ouabain in this assay was 6.0 nM (-0.99).

B. Competition for ³ H-ouabain Binding

Competition for ³ H-ouabain (Amersham Corp. or DuPont-New EnglandNuclear) binding was carried out in 670 μl of binding buffer comprising,in final concentrations, 10 mM TES-Tris (pH 7.4), 0.5 mM EDTA, 5.0 mMMgCl₂ and 5.0 mM Na₂ HPO₄ (hereinafter "binding buffer") and 50 nMouabain containing ³ H-ouabain tracer, and including the unknown sample.Binding was initiated by addition of 0.1-2 μg of purified (Na⁺+K⁺)ATPase and the reaction performed for 2 hr at 37° C. The reactionwas quenched by addition of 2.0 ml aliquots of ice-cold binding buffercontaining 100 μM unlabeled ouabain, followed by vacuum filtration overglass fiber filters (Whatman GF/B). Trapped ³ H-ouabain was determinedby scintillation counting, following 12 hr of soaking the filters in 4.0ml of Beckman Ready Protein ™ scintillation cocktail (Beckman, PaloAlto, Calif.). Nonspecific binding of ³ H-ouabain was estimated byinclusion of excess unlabeled 100 μM ouabain in binding buffer and wasalways less than 2% of the total counts bound. All binding data werecorrected for quench, and specific binding was expressed as pmol/mg ofenzyme protein. Typical values of ³ H-ouabain bound were 75-85 pmol/mgof enzyme protein as compared with phosphorylation levels of 100 pmol ofacid stable ³² P₁ incorporated/mg of enzyme protein. This corresponds toan overall ratio of 0.8 mol of ouabain bound/mol phosphorylation sitesunder type II conditions. The calculated apparent K_(d) value (and Hillcoefficient) for human ouabain in this assay was 7.7 nM (-0.97).

C. Inhibition of (Na⁺ +K⁺)ATPase Activity

The inhibition of (Na⁺ +K⁺)ATPase was carried out as described above.The calculated apparent K_(d) value (and Hill coefficient) for humanouabain in this assay was 13.9 nM (-1.28).

D. Competition for Binding to Antibodies having Binding Specificity forOuabain

The competition for binding to antibodies having binding specificity forouabain was carried out by employing polyclonal antisera havingtiters >10,000 generated against ouabain in rabbits as described inExample 2 below. As discussed in detail in Example 2 below, the antiserashowed no significant cross-reactivity (< about 0.02%) with the commonsteroids present in plasma. A competition ELISA was carried out asfollows:

Microtiter plates were coated with 50 μl of a 1.0 ng/ml solution ofConjugate 4. Following washes with wash buffer comprising 0.9% (w/v)NaCl and 0.5% (v/v) Tween 20 (hereinafter "wash buffer"), residual siteswere blocked with buffer comprising 136 mM NaCl, 1.46 mM KH₂ PO₄, 0.8 mMNa₂ HPO₄, 0.028 mM thimerosal, 0.5% (v/v) Tween 20, and 1.0% (w/v) BSA(pH 7.4). Known amounts of free ouabain or unknown samples wereintroduced followed by the addition of ouabain antisera at an overalldilution of 1:2×10⁶. Incubations at 23° C. for 1 hr were terminated byrepeated washing with wash buffer. Subsequently, a second incubation at≦° C. for 1hr was performed with a 1:10³ dilution of goat anti-rabbitIgG-peroxidase conjugate (Boehringer Mannheim, Indianapolis, Ind.).Following extensive washing with wash buffer, equal parts of 3, 3', 5,5'-tetramethylbenzindine and hydrogen peroxide (Kirkegaard and Perry,Gaitheresburg, Md.) were added and incubated at 23° C. for 15 min. Colordevelopment was terminated by addition of 1.0 M H₃ PO₄ and read at 450nm with a plate reader. Addition of 0.05 to 0.08 pmol of ouabain perwell resulted in 50% displacment of bound ouabain. Each data point isthe mean ± S.E.M. All lines were fitted by nonlinear regression to asigmoidal function. The calculated apparent K_(d) value (and Hillcoefficient) for human ouabain in this assay was 1.6 nM (-0.65).

Inotropic properties of human ouabain were determined in left atria from250 to 300 g guinea pigs mounted in 1.3 ml tissue chambers, between twopairs of platinum hooks; one hook served as an anchor and stimulatingelectrode and the other hook was connected to a force transducer. Atriawere incubated at 37° C. in phosphate buffered saline comprising 130 mMNaCl, 5.4 mM KCl, 1.8 mM MgCl₂, 0.4 mM NaH₂ PO₄, 19 mM NaHCO₃ and 5.4 gof glucose (pH 7.3-7.4), and gassed with 95% O₂ /5% CO₂. Tissues werestimulated at 1 Hz with twice threshold square pulses (0.8 to 1.0 V) of0.7 to 1.2 msec duration. Resting tension was optimized for maximumisometric force generation. Control records were obtained once thephasic force was stabilized. Purified human ouabain was added from astock solution (5.0 μg/ml in water) to give final concentrations of 43to 170 nM. Addition of human ouabain to the bath to give a finalconcentration of 85 nM caused an increase in peak force that reached aplateau in 34 min. Developed force increased further with 170 nM humanouabain to a value of 294 ±28% (N=3 experiments) above the control. Theapparent half-time for washout of the inotropic response to humanouabain was approximately 5 min. For comparison, 200 nM plant ouabainevoked an inotropic response of 311±41% (N=4 experiments) which returnedto normal with a half-time of approximately 5 min upon washout.

Vasoactive properties of human ouabain were determined in rings of aortafrom 250 to 300 g guinea pigs mounted in 1.3 ml tissue chambers, betweentwo pairs of platinum hooks; one hook served as the anchor and the otherhook was connected to a force transducer. The aortic rings wereincubated at 37° C. in phosphate buffered saline. The rings weresubmaximally contracted for 3 to 7 min, every 35 min, with 0.2 or 1.0 μMhistamine. Following three control contractions, the rings were exposedto 170 nM human ouabain for 30 minutes and then recontracted with 0.2 to1.0 μM histamine. Human ouabain had no effect on baseline tension, butincreased the histamine-evoked tension to 150 ±14% of control (N=4experiments). The apparent half-time for washout of the augmentedtension response in the presence of human ouabain was much greater than60 min. For comparison, 200 nM plant ouabain also did not affectbaseline tension, but increased the histamine-evoked tension to 146 ±16%of control (N=4 experiments). The apparent half-time for washout of theaugmented tension response in the presence of plant ouabain was alsomuch greater than 60 min.

EXAMPLE 2 Preparation of Anti-Ouabain Antibodies Preparation ofConjugates

Four different conjugates were prepared. Conjugates 1-3 were used forimmunization of New Zealand White rabbits so as to prepare antibodiesthat bind specifically to ouabain. Conjugate 4 (containing BSA), ratherthan Conjugates 1-3, was used for the ELISA described in both Example 1above, and Example 3 below, to ensure that the antisera would onlyrecognized the ouabain portion of the conjugate.

Conjugate 1 was ovalbumin linked to ouabain using hexane diamine as aspacer. This conjugate was prepared in the following manner: ##STR2##

More specifically, to 2.4 g of hexane diamine free base (20.6 mmol)dissolved in 10 ml water was added 0.53 g of K₂ HPO₄ (2.3 mmol) and thepH of the solution was adjusted to about 6.0 by the addition of 3.0 mlof concentrated aqueous HCl. Then, 350 mg of ovalbumin (Sigma A5503Grade V) was added and dissolved to provide a clear, homogeneoussolution. Next, 1.0 g of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimidehydrochloride (EDAC) (5.22 mmol) was added in a single portion and thesolution stirred at 15° C. for 24 hr. Qualitative measurement of the pHindicated little change during the course of the reaction. The resultingsolution was dialyzed exhaustively against numerous changes of water andlastly against phosphate buffer comprising 1.8 mM K₂ HPO₄ and 63 mM KH₂PO₄ (pH 0.5) (hereinafter "phosphate buffer"). A final volume of 25 mlwas obtained. The major portion of this (18 ml) was lyophilized forstorage, while the remainder (7.0 ml) was carried forward to the ouabaincoupling.

0.5 g of ouabain octahydrate (0.68 mmol) was dissolved in 4.0 ml ofwater and 2.0 ml of acetone by gentle warming. At ambient temperature,0.235 g of NaIO₄ (1.1 mmol) was added in a single portion. A precipitateaccumulated during this reaction. After 2.5 hr, 1.22 g of K₂ HPO₄ (5.0mmol) was added and the reaction was stirred overnight. Then, 85 mg ofmeso-erythritol (0.65 mmol) was added; the reaction left an additional60 min; and the acetone removed on a rotary evaporator. The residue wastaken up in a small quantity of water and added to the above-described7.0 ml ovalbumin solution. After stirring at 0° C. for 90 min, 82 mg ofsolid NaBH₃ CN (1.30 mmol) was added; then 52 mg of solid NaBH₃ CN (0.82mmol) wad added after 2.5 hr; and finally 50 mg of solid NaBH₃ CN (0.80mmol) was added after 5 hr, with the reaction being maintained at 0 °C.throughout. The reaction mixture acquired a cloudy appearance at thistime. It was left at 4° C. for three days, with the appearance ofsignificant quantities of precipitate. The entire reaction mixture wasdialyzed exhaustively against 65 mM potassium phosphate buffer (pH 6.0),concentrated by lyophilization, and then resuspended in 5.0 ml of waterand gently stirred for 2 hr. The precipitate was separated bycentrifugation at 1000×g and the soluble protein passed through a φ4.0cm ×23 cm Sephadex G-25 (20-50 μm) column. The protein fractions werecombined, dialyzed against 10 mM NaCl, and 0.8 μm filtered to provide 27ml of solution containing Conjugate 1.

Conjugate 2 was poly-D-lysine linked directly to ouabain. This conjugatewas prepared in the following manner: ##STR3##

More specifically, 300 mg of ouabain-octahydrate (0.41 mmol) wasdissolved in 3.0 ml water and 2.0 ml acetone by gentle warming. Aftercooling to ambient temperature, 0.215 g of NaIO₄ wad added with therapid formation of a precipitate. During a 5 hr period, 225 mg of K₂HPO₄ was added portionwise (1.0 mmol total). The reaction was thenquenched with 25 mg of meso-erythritol (0.20 mmol) and left at ambienttemperature overnight. The acetone was removed on a rotary evaporatorand the residue taken up in several small volumes of water, total volume4.0 ml. 1.75 ml of this solution was then added to a solution of 36 mgof poly-D-lysine (Sigma P7886: DP 254, M_(r) =53,000 (vis): DP =236,M_(r) =49,000 (lalls)) in 1.0 ml of citrate-phosphate buffer comprising30 mg of citric acid and 50 mg of K₂ HPB₄. A final adjustment of thereaction pH to about 5.0-5.5 was made by the addition of 0.10 ml of 1.0M citric acid. The clear, colorless solution was kept at 0° C. for 4.5hr and then placed at 10° C. overnight. During this time, the reactionacquired a dark yellow-brown color which was discharged instantly by theaddition of a 0.1 ml of a 1.72 M solution of NaBH₃ CN. After 7 hr at 10°C., a faint yellow color appeared; this too was discharged by theaddition of 0.10 ml of a 1.61 M solution of NaBH₃ CN and the reactiononce again kept overnight. The reaction mixture remained colorless andcontained a small quantity of precipitate. It was dialyzed exhaustivelyagainst water at ambient temperature; concentrated by lyophilization;resuspended with 2.5 ml of water, centrifuged at 1000 ×g, and thesupernatant passed through a φ 4.0 cm ×25 Sephadex G-25 (20-50 μm)column. The polypeptide-containing fractions were combined and dialyzedagainst water. After filtration to remove insoluble material, 6.0 ml ofsolution of Conjugate 2 was obtained.

Conjugate 3 was ovalbumin linked to ouabain using succinyl dihydrazideas a spacer. This conjugate was prepared in the following manner:##STR4##

More specifically, 1.0 g of succinyldihydrazide (Aldrich S550-2; 6.8mmol) was dissolved with heating in 14 ml of water. Most, but not all,of the solid remained in solution upon cooling. To this solution wasadded 0.40 g of KH₂ PO₄ and the pH was adjusted to about 5.0 with 0.1 mlof concentrated aqueous HCl. 215 mg of chicken egg ovalbumin and then0.67 g of 1-(3-dimethylaminoprophy)-3-ethyl carbodiimide hydrochloride(3.5 mmol) were added to yield a mostly homogeneous solution. After 20hr at ambient temperature the solution, without precipitate, wasexhaustively dialyzed against water and then finally against acetatebuffer comprising 2.65 g of CH₃ CO₂ Na and 0.70 ml of CH₃ CO₂ H in 500ml of water (pH about 4.5).

Separately, 375 mg of ouabain octahydrate (0.41 mmol) was dissolved in3.0 ml of water and 2.0 ml of acetone by warming. After cooling, 295 mgof NaIO₄ was added. One half hour later, the reaction mixture wasneutralized with 1.0 mmol of CH₃ CO₂ Na and stirred overnight.Meso-erythritol (1.0 mmol) was added as a quench and the acetone removedon a rotary evaporator.

The oxidized ouabain was then added to the ovalbumin at ambienttemperature for 4 hr, and then at 10° C. for 60 hr. No precipitateappeared. After dialysis, the protein was concentrated bylyophilization, resuspended in water, and purified by G-25chromatography. The protein fractions were combined and further dialyzedagainst water. After removing a small portion for UV spectral analysis,the remainder of Conjugate 3 was lyophilized.

Conjugate 4 was BSA linked to ouabain using hexane diamine as a spacer.This conjugate was prepared in the following manner: ##STR5##

More specifically, 3.5 g of hexane diamine free base (30 mmol) wasdissolved in 20 ml of water and the pH adjusted to about 8.0 by theaddition of 11.0 g of solid KH₂ PO₄ (82 mmol). To this clear, lightyellow-colored solution was added 520 mg of BSA (Sigma A7888 RIA grade,about 7.4 μmol) and the protein dissolved by gentle stirring to providea slightly turbid solution. In a single portion was added 1.0 g of1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (5.2 mmol)and the reaction was stirred for 20 hr at ambient temperature. Thereaction was then dialyzed against multiple changed of water and finallyagainst 2.0 liters of water containing 8.0 g of KH₂ PO₄ (58.8 mmol). Theprotein solution was divided into two equal portions. The first wastaken to dryness and the protein stored as the powder. The second wasconcentrated to a volume of about 15 ml by lyophilization and reactedwith the oxidized ouabain as described below.

0.67 g of ouabain octahydrate (0.91 mmol) was dissolved in 4.0 ml ofacetone and 4.0 ml of water with gentle warming. To this was added, atambient temperature, 0.35 g of NaIO₄ (1.7 mmol). Observation of aprecipitate was made within 5 min of the addition; copious solid wasevident within 20 min. An additional 2.0 ml of water was added, withsome dissolution of the precipitate, and the reaction was stirredovernight. Then, 100 mg of meso-tartaric acid was added as a quench andthe acetone removed with a rotary evaporator. The precipitate dissolvedduring this concentration but upon brief standing at 5° C. reappeared.The pH or the oxidized ouabain solution was adjusted to about 4.0 withsolid K₂ HPO₄, combined with the second albumin portion (from above) togive a total volume of 25 ml, and a final adjustment of the pH to about6.0 made with additional K₂ HPO₄. The reagents were stirred together for30 min and then 70 mg of NaBH₃ CN (1.1 mmol) added; followed by adding170 mg of NaBH₃ CN (2.7 mmol) 90 min later. After 17 hr, the reactionmixture was briefly dialyzed against 25 mM Tris buffer (pH 7.9);concentrated by lyophilization; and applied to a φ3.0 cm ×36 cm SephadexG-25 (20-50 μm) column. The protein fractions containing Conjugate 4eluted well separated from the oxidized ouabain, and after overnightdialysis against 25 mM Tris buffer (pH 7.9), were examined by UVdifference spectroscopy.

II. Immunization and Collection of Antisera

Antibodies which specifically bind to ouabain were prepared as follows:

9 New Zealand White rabbits were immunized using 1.0 mg/ml of Conjugate1 dissolved in an emulsion comprising 0.5 ml of saline and 0.5 ml ofcomplete Freunds adjuvant. This emulsion was injected intradermally atten sites distributed along each side of the spinal column on the shavedback of the rabbits using 0.1 ml of emulsion per site.

Four weeks after the first immunization, the rabbits were boosted byadministering 1.0 mg/ml of Conjugate 2 dissolved in an emulsioncomprising 0.5 ml of saline and 0.5 ml of incomplete Freunds adjuvant.This emulsion was injected intradermally at ten sites distributed alongeach side of the spinal column on the shaved back of the rabbits using0.1 ml of emulsion per site.

Four weeks after the second immunization, the rabbits were boosted byadministering 1.0 mg/ml of Conjugate 3 dissolved in an emulsioncomprising 0.5 ml of saline and 0.5 ml of incomplete Freunds adjuvant.This emulsion was injected intradermally ten times at five sites alongeach side of the spinal column on the shaved back of the rabbits using0.1 ml of emulsion per site.

In each immunization, the conjugates shared a common ouabain moietywhile the carrier protein or spacer group used to link ouabain to thecarrier was varied. This protocol was designed to make ouabain theemphasis of the antigenic response and to de-emphasize any response tothe carrier protein or spacer group used.

Polyclonal anti-ouabain antisera was obtained from the immunized rabbitsby collecting blood from the central ear artery, allowing the blood toclot for 2 hr at room temperature, centrifuging the clotted blood at1000 ×g for 10 min, and removing the polyclonal anti-ouabain antisera.

The polyclonal antisera was extensively characterized by testing thedegree to which it cross-reacted with (1) other substances known to bepresent in human body fluids and tissues, and (2) cardiotonic steroidsclosely related to ouabain but not known to be present in human bodyfluids and tissues. These substances and their cross-reactivities(relative to ouabain) are set forth in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Substances Known to Be Present                                                in Human Body Fluids and Tissues                                              Aldosterone            0.012%                                                 Atrial natriuretic peptide                                                                           None detected                                          Angiotensin I          None detected                                          Angiotensin II         None detected                                          Arginine vasopressin   None detected                                          β-estradiol       None detected                                          Chlormadinone acetate  None detected                                          Cholesterol            None detected                                          Citrate                None detected                                          Corticosterone         0.0035%                                                Deoxycorticosterone    0.0046%                                                Dehydroisoandrosterone None detected                                          Dehydroisoandrosterone-3-sulfate                                                                     None detected                                          Hydrocortisone         None detected                                          Progesterone           0.0011%                                                Renin                  None detected                                          Taurocholic acid       None detected                                          Testosterone           0.0042                                                 Cardiotonic Steroids Not Known                                                to be Present in Human Body                                                   Fluids and Tissues                                                            Digoxin                5.2%                                                   Digitoxin              28%                                                    Dihydroouabain         0.16%                                                  Lanatoside C           2.49%                                                  Ouabagenin             40%                                                    Strophanthidin         66%                                                    ______________________________________                                    

It is apparent from the results shown in Table 1 above that thisantisera is highly specific for ouabain and other closely relatedcardiotonic steroids, such as digitoxin, ouabagenin and strophanthidin.None of the compounds present in human body fluids or tissuescross-reacted significantly. The dissociation constant of the antibodiesis in the range of 2.0 to 4.0 nM of ouabain and is thus of highaffinity.

EXAMPLE 3 Immunological Assay I. Enrichment of Samples

In order to effect enrichment of the plasma, the plasma sample wasdiluted at least 1:5 (w/v or v/v) with 0.1% (w/v) TFA in water. Then,the diluted sample was passed through a 0.5 g Analytichem InternationalC₁₈ column which had previously been wetted with 5.0 ml of 100% (v/v)acetonitrile, followed by washing with 10 ml of water. Once the samplewas applied to the column, the column was washed twice with 3.0 ml ofwater to remove any materials which did not bind. A single fraction waseluted and collected by washing the column with 3.0 ml of 25% (v/v)acetonitrile. This fraction was dried and redissolved in assay buffer.The resulting solution was used for determination of plasma levels ofhuman ouabain as described below.

II. Assay of Samples

The anti-ouabain antisera obtained in Example 2 was utilized in acompetition ELISA to measure plasma levels of human ouabain usingouabain as a standard as described below.

A. Preparation of Support

96-well EIA microtiter plates (Costar, Mass.) were coated with Conjugate4 by adding 50 μl of a solution containing 50 pg of Conjugate 4dissolved in 0.05 M carbonate-bicarbonate buffer (pH 9.8), and incubatedovernight at 4° C. After incubation, any Conjugate 4 which had notadhered to the microtiter plate was removed by washing the wells 3 timeswith 200 μl/well of rinse solution comprising 0.05% (v/v) Tween 20 and150 mM NaCl (hereinafter "rinse solution"). This removed the excessunbound Conjugate 4 leaving only that bound to the EIA plate.

The remaining non-specific binding sites in each well on the plates wereblocked by adding 100 μl of assay buffer to each well and incubating theresulting microtiter plates at room temperature for at least 1 hr or at4° C. for at least overnight. Then, the assay buffer was removed bywashing the wells with 200 μl/well of rinse solution.

B. Preparation of Standard

A ouabain standard curve was prepared by adding 25 μl of differentconcentrations of ouabain (Sigma) dissolved in assay buffer, rangingfrom 0.025 pmole/25 μl to 12.8 pmole/25 μl, to different wells of themicrotiter plate.

C. Unknown Sample

The level of human ouabain in the unknown sample was measured by adding25 μl of different dilutions of an unknown sample to different wells ofthe microtiter plate.

D. Initiating Assay

A competition ELISA was initiated by adding 25 μl of anti-ouabainantisera (diluted 1:30,000 or more with assay buffer) to each well andincubating for 1 hr in a shaking bath at room temperature.

The anti-ouabain antibody recognizes both the ouabain portion ofConjugate 4 (attached to the microtiter plate) as well as the ouabainstandard and human ouabain. The amount of anti-ouabain antibody thatbinds to Conjugate 4 which is bound to the microtiter plate is inverselyproportional to the amount of ouabain standard or human ouabain.

E. Development of Assay

After 1 hr of incubation, each well was washed 3 times with 200 μl ofrinse solution. Into each well was added 50 μl of goat anti-rabbit IgGantibody-peroxidase conjugate (Boehringer Mannheim Biochemicals) diluted1:1000 with assay buffer. The microtiter plates were then incubated in ashaking bath with this second antibody for 1 hr at room temperature.

The second incubation step allows the anti-IgG antibody to bind to therabbit anti-ouabain antibody which is bound to the well on themicrotiter plate. Therefore, the amount of goat anti-rabbit IgG whichwill bind is directly proportional to the amount of rabbit anti-ouabainantibody which was bound in the first incubation. After this secondincubation, each well was washed 4 times with 200 μl of rinse solutionto remove any free goat anti-rabbit IgG antibody-peroxidase conjugate.

The amount of anti-rabbit IgG antibody-peroxidase conjugate remainingbound in each well was measured by adding 50 μl of a peroxidasesubstrate consisting of 1 part Kirkegaard and Perry (Gaithersburg, Md.)3, 3', 5, 5'-tetramethylbenzidine Solution A reagent and 1 partKirkegaard and Perry hydrogen peroxide Solution B reagent. Themicrotiter plates were then shaken at room temperature and the resultingblue color was allowed to develop until an approximate absorbance of0.75 O.D. units was obtained at 650 nm. At this point, the reaction wasterminated by adding 50 μl of 1.0 M H₃ PO₄. The addition of H₃ PO₄causes an increase in color intensity and changes the reagent color fromblue to yellow. Therefore, the OD was measured at 450 nm. The wellsdeveloping more color contained more goat anti-rabbit IgGantibody-peroxidase conjugate, and thus contained more rabbitanti-ouabain antibody. This means that they contained less ouabainstandard or human ouabain in the original incubation mixture because thebinding of anti-ouabain antibody to Conjugate 4 bound to the wells ineach microtiter plate is inversely proportional to the ouabain standardor human ouabain concentration during the initial incubation of thecompetition ELISA. The wells developing less color contained little goatanti-rabbit IgG antibody-peroxidase conjugate. Therefore, they containedlittle rabbit anti-ouabain antibody. This means that these samplescontained higher levels of ouabain or human ouabain in the originalincubation mixture.

A standard curve was prepared by plotting observed OD₄₅₀ versus theknown amount of ouabain added to the standard curve wells of theoriginal incubation mixture. By comparing the OD₄₅₀ obtained for humanouabain samples with the amount of ouabain on the standard curverequired to give the same OD, unknown human ouabain levels weredetermined.

This assay technique can be used to measure levels of human ouabain in asubject afflicted with pre-hypertension, hypertension (Example 4 below)and white-coat hypertension, in a subject at risk to develop renalfailure (Example 4 below), in a subject at risk to develop congestivecardiomyopathy (Example 5 below), in salt-sensitive subjects, and insubjects afflicted with adenomas and endocrine cell hyperplasias. In allinstances, except the congestive cardiomyopathy, elevated levels, i.e.,above the normal range, of human ouabain are indicative of theaforementioned conditions. Levels of human ouabain are reduced incongestive cardiomyopathy and in otherwise normal individuals who are atrisk for future development of cardiomyopathy.

EXAMPLE 4 Measurement of Ouabain Levels in Rats with Induced RenalDisease and Hypertension

Three groups of rats were studied: (i) Normal 300 g Sprague Dawley malerats; (ii) 300 g Sprague Dawley male rats subjected to unilateralnephrectomy (removal of one kidney) and administered 0.9% (w/v)NaCl-drinking water for 2 weeks to simulate renal disease; and (iii) 300g Sprague Dawley male unilateral nephrectomized rats administered 0.9%(w/v) NaCl-drinking water, and treated with a silastic implantimpregnated with 100 mg/kg deoxycorticosterone acetate for 2 weeks toinduce adrenocorticoid-induced hypertension. After 2 weeks of theappropriate treatment, the mean arterial blood pressure in each rat wasmeasured by the tail cuff method (Cimini, C.M. et al, Lab, Animal Sci.,35:412-416 (1985); tail cuff purchased from IITC, Landing, N.J.). Trunkblood or venous blood was removed from unstressed rats and was collectedin chilled tubes containing (in final concentrations) 2.8 mM Na₂ EDTA,3.4 mM Na₂ EGTA and 2.6 mM glutathione. The blood was continuouslystirred to ensure uniform anti-coagulation. Then, the blood wascentrifuged at 8000 to 10,000 ×g for 10 min at room temperature. Theplasma was removed in 5.0 ml aliquots and stored at -20° to -70° C. forup to 5 to 6 months. The fresh or thawed plasmas were enriched forouabain as described in Example 3, Section I, and the resulting enrichedsamples were assayed for levels of ouabain using the ELISA described inExample 3, Section II. The results obtained are shown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                                    Mean Arterial                                                                 Blood Pressure Ouabain Levels                                     Group       (mm Hg)        (pmol/liter)                                       ______________________________________                                        Normal      99.8 ± 1.5 (N = 6)*                                                                        66 ± 6 (N = 8)                                 Renal Disease                                                                              113 ± 4 (N = 6)                                                                          105 ± 43 (N = 6)                                Hypertension                                                                               156 ± 12 (N = 5)                                                                         975 ± 79 (N = 5)                                ______________________________________                                         *Data are the means ± standard errors for N rats.                     

The results in Table 2 above demonstrate that rats with renal diseasehave elevated levels of ouabain with only a small increase in bloodpressure, whereas hypertensive rats have elevated levels of ouabain andan increased blood pressure. These rat models for renal disease andhypertension, which are well-known to be correlatable to renal diseaseand hypertension in humans, demonstrate that levels of human ouabain canbe employed to diagnose renal disease and hypertension in humans and tomonitor therapies for renal disease and hypertension in humans.

EXAMPLE 5 Measurement of Ouabain Levels in Humans with Cardiomyopathy

Two groups of humans were studied: (i) normal subjects; and (ii)subjects with untreated left ventricular cardiomyopathy. Blood wasremoved from these subjects by venipuncture and collected in chilledtubes as described in Example 4. The plasmas were separated as describedin Example 4 and then enriched for human ouabain as described in Example3, Section I, and the resulting enriched samples were assayed for levelsof human ouabain using the ELISA described in Example 3, Section II. Theresults obtained are shown in Table 3 below.

                  TABLE 3                                                         ______________________________________                                                            Ouabain Levels                                            Group               (pmol/liter)                                              ______________________________________                                        Normal              53 ± 8 (N = 7)*                                        Cardiomyopathy      24 ± 8 (N = 4)                                         ______________________________________                                         *Data are the means ± standard errors for N subjects.                 

The results in Table 3 above demonstrate that levels of human ouabainare reduced in patients with untreated cardiomyopathy and thus that thelevels of human ouabain can be employed to determine subjects at risk todevelop congestive cardiomyopathy and to monitor therapy for congestivecardiomyopathy.

While this invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes can be made therein without departing from thespirit and scope thereof.

We claim:
 1. A method for diagnosing pre-hypertension comprising thesteps of:(1) obtaining a body fluid or tissue sample from a subject withnormal blood pressure; (2) enriching the resulting body fluid or tissuesample obtained in step (1) for human ouabain; (3) using an antibodyhaving binding specificity for ouabain, immunologically measuring thelevel of human ouabain in the resulting enriched body fluid or tissuesample of step (2); and (4) comparing the level of human ouabainobtained in step (3) with an ouabain standard so as to detect a normalor elevated level of human ouabain in the subject and to diagnose theabsence or presence of pre-hypertension, respectively.
 2. The method asclaimed in claim 1, wherein said body fluid is selected from the groupconsisting of plasma, cerebrospinal fluid, saliva, semen, sweat, urineand amniotic fluid.
 3. The method as claimed in claim 2, wherein saidbody fluid is plasma.
 4. The method as claimed in claim 1, wherein saidtissue sample is selected from the group consisting of adrenal tissue,red blood cells, lymphocytes, and platelets.
 5. The method as claimed inclaim 4, wherein said tissue sample is red blood cells.
 6. The method asclaimed in claim 1, wherein said antibody has a dissociation constant onthe order of about 7.0 nM or less of ouabain, and a cross-reactivity forsteroids present in human plasma on the order of about 1.0% or lesscross-reactivity
 7. The method as claimed in claim 6, wherein saidantibody has a dissociation constant on the order of about 0.5 to 7.0 nMof ouabain, and a cross-reactivity for steroids present in human plasmaon the order of about 0.02 to 0.001% cross-reactivity.
 8. The method asclaimed in claim 1, wherein said elevated level of human ouabain isabout 100 to 4000 pmol/liter of plasma.
 9. A method for diagnosinghypertension comprising the steps of:(1) obtaining a body fluid ortissue sample from a subject with high blood pressure; (2) enriching theresulting body fluid or tissue sample obtained in step (1) for humanouabain; (3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and (4) comparing thelevel of human ouabain obtained in step (3) with an ouabain standard soas to detect a normal or elevated level of human ouabain in the subjectand to diagnose the absence or presence of hypertension, respectively.10. The method as claimed in claim 9, wherein said body fluid isselected from the group consisting of plasma, cerebrospinal fluid,saliva, semen, sweat, urine and amniotic fluid.
 11. The method asclaimed in claim 10, wherein said body fluid is plasma.
 12. The methodas claimed in claim 9, wherein said tissue sample is selected from thegroup consisting of adrenal tissue, red blood cells, lymphocytes, andplatelets.
 13. The method as claimed in claim 12, wherein said tissuesample is red blood cells.
 14. The method as claimed in claim 9, whereinsaid antibody has a dissociation constant on the order of about 7.0 nMor less of ouabain, and a cross-reactivity for steroids present in humanplasma on the order of about 1.0% or less cross-reactivity
 15. Themethod as claimed in claim 14, wherein said antibody has a dissociationconstant on the order of about 0.5 to 7.0 nM of ouabain, and across-reactivity for steroids present in human plasma on the order ofabout 0.02 to 0.001% cross-reactivity.
 16. The method as claimed inclaim 9, wherein said elevated level of human ouabain is about 250 to2000 pmol/liter of plasma.
 17. The method as claimed in claim 9, whereinsaid hypertension is selected from the group consisting of essentialhypertension, adrenocorticoid-induced hypertension, pregnancy-inducedhypertension, renal hypertension and pheochromocytoma.
 18. A method fordiagnosing white-coat hypertension comprising the steps of:(1) obtaininga body fluid or tissue sample from a subject with high blood pressure;(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain; (3) using an antibody having binding specificityfor ouabain, immunologically measuring the level of human ouabain in theresulting enriched body fluid or tissue sample of step (2); and (4)comparing the level of human ouabain obtained in step (3) with anouabain standard so as to detect a normal or elevated level of humanouabain in the subject and to diagnose the presence or absence ofwhite-coat hypertension, respectively.
 19. The method as claimed inclaim 18, wherein said body fluid is selected from the group consistingof plasma, cerebrospinal fluid, saliva, semen, sweat, urine and amnioticfluid.
 20. The method as claimed in claim 19, wherein said body fluid isplasma.
 21. The method as claimed in claim 18, wherein said tissuesample is selected from the group consisting of adrenal tissue, redblood cells, lymphocytes, and platelets.
 22. The method as claimed inclaim 21, wherein said tissue sample is red blood cells.
 23. The methodas claimed in claim 18, wherein said antibody has a dissociationconstant on the order of about 7.0 nM or less of ouabain, and across-reactivity for steroids present in human plasma on the order ofabout 1.0% or less cross-reactivity
 24. The method as claimed in claim23, wherein said antibody has a dissociation constant on the order ofabout 0.5 to 7.0 nM of ouabain; and a cross-reactivity for steroidspresent in human plasma on the order of about 0.02 to 0.001%cross-reactivity.
 25. The method as claimed in claim 18, wherein saidnormal level of human ouabain is about 40 to 95 pmol/liter of plasma.26. A method for determining subjects at risk to develop congestivecardiomyopathy comprising the steps of:(1) obtaining a body fluid ortissue sample from a subject with cardiomyopathy; (2) enriching theresulting body fluid or tissue sample obtained in step (1) for humanouabain; (3) using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and (4) comparing thelevel of human ouabain measured in step (3) with a ouabain standard soas to detect a normal or reduced level of human ouabain in the subjectand to diagnose the absence or presence of the risk of developingcongestive cardiomyopathy, respectively.
 27. The method as claimed inclaim 26, wherein said body fluid is selected from the group consistingof plasma, cerebrospinal fluid, saliva, semen, sweat, urine and amnioticfluid.
 28. The method as claimed in claim 27, wherein said body fluid isplasma.
 29. The method as claimed in claim 26, wherein said tissuesample is selected from the group consisting of adrenal tissue, redblood cells, lymphocytes, and platelets.
 30. The method as claimed inclaim 29, wherein said tissue sample is red blood cells.
 31. The methodas claimed in claim 26, wherein said antibody has a dissociationconstant on the order of about 7.0 nM or less of ouabain, and across-reactivity for steroids present in human plasma on the order ofabout 1.0% or less cross-reactivity
 32. The method as claimed in claim31, wherein said antibody has a dissociation constant on the order ofabout 0.5 to 7.0 nM of ouabain; and a cross-reactivity for steroidspresent in human plasma on the order of about 0.02 to 0.001%cross-reactivity.
 33. The method as claimed in claim 26, wherein saidreduced level of human ouabain is about 10 to 35 pmol/liter of plasma.34. The method as claimed in claim 26, wherein said cardiomyopathy isselected from the group consisting of ischemic heart disease, familialcardiomyopathy, alcoholic cardiomyopathy, peripartum cardiomyopathy,endocardial fibroelastosis, postcarditic cardiomyopathy, hypertensivecardiomyopathy, idiopathic cardiomyopathy, and secondary forms ofmyocardial involvement in connective tissue diseases and neuromusculardiseases.
 35. A method for determining subjects at risk to develop renalfailure comprising the steps of:(1) obtaining a body fluid or tissuesample from a subject with renal disease; (2) enriching the resultingbody fluid or tissue sample obtained in step (1) for human ouabain; (3)using an antibody having binding specificity for ouabain,immunologically measuring the level of human ouabain in the resultingenriched body fluid or tissue sample of step (2); and (4) comparing thelevel measured in (3) with an ouabain standard so as to detect a normalor elevated level of human ouabain in the subject and to diagnose theabsence or presence of the risk of developing renal failure,respectively.
 36. The method as claimed in claim 35, wherein said bodyfluid is selected from the group consisting of plasma, cerebrospinalfluid, saliva, semen, sweat, urine and amniotic fluid.
 37. The method asclaimed in claim 36, wherein said body fluid is plasma.
 38. The methodas claimed in claim 35, wherein said tissue sample is selected from thegroup consisting of adrenal tissue, red blood cells, lymphocytes, andplatelets.
 39. The method as claimed in claim 38, wherein said tissuesample is red blood cells.
 40. The method as claimed in claim 35,wherein said antibody has a dissociation constant on the order of about7.0 nM or less of ouabain, and a cross-reactivity for steroids presentin human plasma on the order of about 1.0% or less cross-reactivity 41.The method as claimed in claim 40, wherein said antibody has adissociation constant on the order of about 0.5 to 7.0 nM of ouabain;and a cross-reactivity for steroids present in human plasma on the orderof about 0.02 to 0.001% cross-reactivity.
 42. The method as claimed inclaim 35, wherein said elevated level of human ouabain is about 100 to4000 pmol/liter of plasma.
 43. The method as claimed in claim 35,wherein said renal disease is selected from the group consisting ofglomerulonephritis, diabetic nephropathy, tubulointerstital disease,polycystic renal disease and nephrosclerosis.
 44. A method fordetermining the salt-sensitivity of a subject at risk to develophypertension or a hypertensive subject comprising:(1) obtaining a bodyfluid or tissue sample from a subject at risk to develop hypertension ora hypertensive subject, respectively; (2) enriching the resulting bodyfluid or tissue sample obtained in step (1) for human ouabain; (3) usingan antibody having binding specificity for ouabain, immunologicallymeasuring the level of human ouabain in the resulting enriched bodyfluid or tissue sample of step (2); (4) comparing the level of humanouabain measured in step (3) with an ouabain standard so as to detect anormal or elevated level of human ouabain in the subject; and (5)repeating steps (1)-(4) at suitable time intervals after the initiationof a low-salt or high-salt diet and measuring the level of human ouabainat each time interval so as to determined the salt-sensitivity of saidsubject, wherein when the level of human ouabain is elevated and theelevated level of human ouabain is due to a change from a low-salt to ahigh-salt diet, such is indicative of salt-sensitivity.
 45. The methodas claimed in claim 44, wherein said body fluid is selected from thegroup consisting of plasma, cerebrospinal fluid, saliva, semen, sweat,urine and amniotic fluid.
 46. The method as claimed in claim 45, whereinsaid body fluid is plasma.
 47. The method as claimed in claim 44,wherein said tissue sample is selected from the group consisting ofadrenal tissue, red blood cells, lymphocytes, and platelets.
 48. Themethod as claimed in claim 47, wherein said tissue sample is red bloodcells.
 49. The method as claimed in claim 44, wherein said antibody hasa dissociation constant on the order of about 7.0 nM or less of ouabain,and a cross-reactivity for steroids present in human plasma on the orderof about 1.0% or less cross-reactivity
 50. The method as claimed inclaim 49, wherein said antibody has a dissociation constant on the orderof about 0.5 to 7.0 nM of ouabain; and a cross-reactivity for steroidspresent in human plasma on the order of about 0.02 to 0.001%cross-reactivity.
 51. The method as claimed in claim 44, wherein whensaid said subject is a subject at risk to develop hypertension, saidelevated level of human ouabain is about a 2-fold or greater increase inhuman ouabain; and when said subject is a hypertensive subject, saidelevated level of human ouabain is about 250 to 2000 pmol/liter ofplasma.
 52. The method as claimed in claim 44, wherein said subject atrisk to develop hypertension is selected from the group consisting of asubject who has one or both parents that are hypertensive, an overweightsubject, a subject with high normal blood pressure and a subject withrenal disease.
 53. The method as claimed in claim 44, wherein saidlow-salt diet is an intake of about 60 meq of sodium per day or less.54. The method as claimed in claim 53, wherein said low-salt diet is anintake of about 40 to 60 meq of sodium per day.
 55. The method asclaimed in claim 44, wherein said high-salt diet is an intake of about200 meq of sodium per day or greater.
 56. The method as claimed in claim55, wherein said high-salt diet is an intake of about 200 to 250 meq ofsodium per day.
 57. A method for diagnosing adenomas or endocrine cellhyperplasias comprising the steps of:(1) obtaining a body fluid ortissue sample from a subject; (2) enriching the resulting body fluid ortissue sample obtained in step (1) for human ouabain; (3) using anantibody having binding specificity for ouabain, immunologicallymeasuring the level of human ouabain in the resulting enriched bodyfluid or tissue sample of step (2); (4) comparing the level of humanouabain measured in step (3) with an ouabain standard so as to detect anormal or elevated level of human ouabain in the subject; and (5)repeating steps (1)-(4) at suitable time intervals after the initiationof a low-salt or high-salt diet and measuring the level of human ouabainat each time interval so as to determine the presence of adenomas orendocrine cell hyperplasias in said subject, wherein when the level ofhuman ouabain is elevated and the elevated level of human ouabain is notdue to a change from a low-salt to a high-salt diet, such is indicativeof adenomas or hyperplasias.
 58. The method as claimed in claim 57,wherein said body fluid is selected from the group consisting of plasma,cerebrospinal fluid, saliva, semen, sweat, urine and amniotic fluid. 59.The method as claimed in claim 58, wherein said body fluid is plasma.60. The method as claimed in claim 57, wherein said tissue sample isselected from the group consisting of adrenal tissue, red blood cells,lymphocytes, and platelets.
 61. The method as claimed in claim 60,wherein said tissue sample is red blood cells.
 62. The method as claimedin claim 57, wherein said antibody has a dissociation constant on theorder of about 7.0 nM or less of ouabain, and a cross-reactivity forsteroids present in human plasma on the order of about 1.0% or lesscross-reactivity
 63. The method as claimed in claim 62, wherein saidantibody has a dissociation constant on the order of about 0.5 to 7.0 nMof ouabain; and a cross-reactivity for steroids present in human plasmaon the order of about 0.02 to 0.001% cross-reactivity.
 64. The method asclaimed in claim 57, wherein said elevated level of human ouabain isabout 100 to 4000 pmol/liter of plasma.
 65. The method as claimed inclaim 57, wherein said adenomas and endocrine cell hyperplasias areselected from the group consisting of ovarian, adrenal, pituitary andtesticular adenomas and endocrine cell hyperplasias and adenomas andhyperplasias of human ouabain secreting endocrine cells.
 66. The methodas claimed in claim 57, wherein said low-salt diet is an intake of about60 meq of sodium per day or less.
 67. The method as claimed in claim 65,wherein said low-salt diet is an intake of about 40 to 60 meq of sodiumper day.
 68. The method as claimed in claim 57, wherein said high-saltdiet is an intake of about 200 meq of sodium per day or greater.
 69. Themethod as claimed in claim 68, wherein said high-salt diet is an intakeof about 200 to 250 meq of sodium per day.
 70. A method for monitoringhypertension therapy comprising the steps of:(1) obtaining a body fluidor tissue sample from a subject diagnosed as afflicted withhypertension; (2) enriching the resulting body fluid or tissue samplesobtained in step (1) for human ouabain; (3) using an antibody havingbinding specificity for ouabain, immunologically measuring the level ofhuman ouabain in the resulting enriched body fluid or tissue sample ofstep (2); and (4) repeating steps (1)-(3) at suitable time intervalsafter the initiation of hypertension therapy and measuring the level ofhuman ouabain at each time interval so as to monitor the effect of saidhypertension therapy, wherein a reduced level of human ouabain over thecourse of hypertension therapy is indicative of effective hypertensiontherapy.
 71. The method as claimed in claim 70, wherein said body fluidis selected from the group consisting of plasma, cerebrospinal fluid,saliva, semen, sweat, urine and amniotic fluid.
 72. The method asclaimed in claim 71, wherein said body fluid is plasma.
 73. The methodas claimed in claim 70, wherein said tissue sample is selected from thegroup consisting of adrenal tissue, red blood cells, lymphocytes, andplatelets.
 74. The method as claimed in claim 73, wherein said tissuesample is red blood cells.
 75. The method as claimed in claim 70,wherein said antibody has a dissociation constant on the order of about7.0 nM or less of ouabain, and a cross-reactivity for steroids presentin human plasma on the order of about 1.0% or less cross-reactivity 76.The method as claimed in claim 75, wherein said antibody has adissociation constant on the order of about 0.5 to 7.0 nM of ouabain;and a cross-reactivity for steroids present in human plasma on the orderof about 0.02 to 0.001% cross-reactivity.
 77. The method as claimed inclaim 70, wherein said hypertension therapy is selected from the groupconsisting of a low-salt diet, a diuretic, exercise, cessation ofsmoking, cessation of alcohol intake, administration of an α-blocker,administration of a β-blocker, administration of a converting enzymeinhibitor, administration of a calcium channel blocker and a combinationthereof.
 78. A method for monitoring congestive cardiomyopathy therapycomprising the steps of:(1) obtaining a body fluid or tissue sample froma subject diagnosed as afflicted with congestive cardiomyopathy; (2)enriching the resulting body fluid or tissue sample obtained in step (1)for human ouabain; (3) using an antibody having binding specificity forouabain, immunologically measuring the level of human ouabain in theresulting enriched body fluid or tissue sample of step (2); and (4)repeating steps (1)-(3) at suitable time intervals after the initiationof congestive cardiomyopathy therapy and measuring the level of humanouabain at each time interval so as to monitor the effect of saidcongestive cardiomyopathy therapy, wherein an increasing level of humanouabain over the course of congestive cardiomyopathy therapy isindicative of effective congestive cardiomyopathy therapy.
 79. Themethod as claimed in claim 78, wherein said body fluid is selected fromthe group consisting of plasma, cerebrospinal fluid, saliva, semen,sweat, urine and amniotic fluid.
 80. The method as claimed in claim 79,wherein said body fluid is plasma.
 81. The method as claimed in claim80, wherein said tissue sample is selected from the group consisting ofadrenal tissue, red blood cells, lymphocytes, and platelets.
 82. Themethod as claimed in claim 81, wherein said tissue sample is red bloodcells.
 83. The method as claimed in claim 78, wherein said antibody hasa dissociation constant on the order of about 7.0 nM or less of ouabain,and a cross-reactivity for steroids present in human plasma on the orderof about 1.0% or less cross-reactivity
 84. The method as claimed inclaim 83, wherein said antibody has a dissociation constant on the orderof about 0.5 to 7.0 nM of ouabain; and a cross-reactivity for steroidspresent in human plasma on the order of about 0.02 to 0.001%cross-reactivity.
 85. A method as claimed in claim 78, wherein saidcongestive cardiomyopathy therapy is selected from the group consistingof a low-salt diet, administration of a diuretic, administration of acardiotonic steroid, administration of a vasodilator, administration ofa converting enzyme inhibitor, administration of a phosphodiesteraseinhibitor and a combination thereof.
 86. A method for monitoring renalfailure therapy comprising the steps of:(1) obtaining a body fluid ortissue sample from a subject diagnosed as afflicted with renal failure;(2) enriching the resulting body fluid or tissue sample obtained in step(1) for human ouabain; (3) using an antibody having binding specificityfor ouabain, immunologically measuring the level of human ouabain in theresulting enriched body fluid or tissue sample of step (2); and (4)repeating steps (1)-(3) at suitable time intervals after the initiationof renal failure therapy and measuring the level of human ouabain ateach time interval so as to monitor the effect of said renal failuretherapy, wherein a decreasing level of human ouabain over the course ofrenal failure therapy is indicative of effective renal failure therapy.87. The method as claimed in claim 86, wherein said body fluid isselected from the group consisting of plasma, cerebrospinal fluid,saliva, semen, sweat, urine and amniotic fluid.
 88. The method asclaimed in claim 87, wherein said body fluid is plasma.
 89. The methodas claimed in claim 86, wherein said tissue sample is selected from thegroup consisting of adrenal tissue, red blood cells, lymphocytes, andplatelets.
 90. The method as claimed in claim 89, wherein said tissuesample is red blood cells.
 91. The method as claimed in claim 86,wherein said antibody has a dissociation constant on the order of about7.0 nM or less of ouabain, and a cross-reactivity for steroids presentin human plasma on the order of about 1.0% or less cross-reactivity 92.The method as claimed in claim 91, wherein said antibody has adissociation constant on the order of about 0.5 to 7.0 nM of ouabain;and a cross-reactivity for steroids present in human plasma on the orderof about 0.02 to 0.001% cross-reactivity.
 93. The method as claimed inclaim 86, wherein said renal failure therapy is selected from the groupconsisting of dietary potassium restriction, dietary protein restrictionand dialysis.
 94. A method for monitoring adenoma or endocrine cellhyperplasia therapy comprising the steps of:(1) obtaining a body fluidor tissue sample from a subject diagnosed as afflicted with adenomas orendocrine cell hyperplasias; (2) enriching the resulting body fluid ortissues sample obtained in step (1) for human ouabain; (3) using anantibody having binding specificity for ouabain, immunologicallymeasuring the level of human ouabain in the resulting enriched bodyfluid or tissue sample of step (2); and (4) repeating steps (1)-(3) atsuitable time intervals after the initiation of adenoma or endocrinecell hyperplasia therapy and measuring the level of human ouabain ateach time interval so as to monitor the effect of said adenoma orendocrine cell hyperplasia therapy, wherein a decreasing level of humanouabain over the course of adenoma or endocrine cell hyperplasia therapyis indicative of effective adenoma or endocrine cell hyperplasiatherapy.
 95. The method as claimed in claim 94, wherein said body fluidis selected from the group consisting of plasma, cerebrospinal fluid,saliva, semen, sweat, urine and amniotic fluid.
 96. The method asclaimed in claim 95, wherein said body fluid is plasma.
 97. The methodas claimed in claim 94, wherein said tissue sample is selected from thegroup consisting of adrenal tissue, red blood cells, lymphocytes, andplatelets.
 98. The method as claimed in claim 97, wherein said tissuesample is red blood cells.
 99. The method as claimed in claim 94,wherein said antibody has a dissociation constant on the order of about7.0 nM or less of ouabain, and a cross-reactivity for steroids presentin human plasma on the order of about 1.0% or less cross-reactivity 100.The method as claimed in claim 99, wherein said antibody has adissociation constant on the order of about 0.5 to 7.0 nM of ouabain;and a cross-reactivity for steroids present in human plasma on the orderof about 0.02 to 0.001% cross-reactivity.
 101. The method as claimed inclaim 94, wherein said adenoma or endocrine cell hyperplasia therapy isselected from the group consisting of the use of spironolactone andsurgery.